Compositions and methods for treating ovarian tumors

ABSTRACT

Described herein are compositions and methods of using single-cell RNA-sequencing to identify treatment resistance in patients with ovarian cancer. Also, described herein are compositions and methods for treatment targeting resistance in patients with ovarian cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.62/479,885, filed Mar. 31, 2017 and 62/565,470, filed Sep. 29, 2017. Theentire contents of the above-identified applications are hereby fullyincorporated herein by reference.

TECHNICAL FIELD

The subject matter disclosed herein is generally directed tocompositions and methods for treating ovarian tumors by targetingJAK/STAT signaling.

BACKGROUND

There are over 22,000 new cases of ovarian cancer diagnosed in theUnited States every year. Up to 75% of women with advanced ovariancancer relapse within two years after successfully responding to initialchemotherapy treatment. Accordingly, one of the main obstacles toovercome in improving outcomes among ovarian cancer patients isresistance to chemotherapy. As such, prior to the invention describedherein, there was a pressing need to develop methods to identify geneticprofiles of treatment-resistant ovarian cancer to identify therapeutictargets for recurrent disease.

SUMMARY

Described herein is the use of signal transducer and activator oftranscription 3 (STAT3) inhibitors, as a single drug or as an adjuvant,to treat ovarian tumors. Specifically, provided are methods for treatingor preventing a gynecological tumor in a subject, e.g., a human subject,comprising identifying a subject with a gynecological tumor,administering to the subject a therapeutically effective amount of aSTAT3 activity inhibitor, thereby treating or preventing thegynecological tumor in the subject. An exemplary gynecological tumorcomprises an ovarian tumor.

In one aspect, the subject is identified as having elevated STAT3activity, or the subject is identified as in need of inhibiting STAT3activity. For example, the STAT3 activity is selected from the groupconsisting of STAT3 phosphorylation, STAT3 dimerization, STAT3 bindingto a polynucleotide comprising a STAT3 binding site, STAT3 binding togenomic DNA, activation of a STAT3 responsive gene and STAT3 nucleartranslocation.

In one case, the STAT3 activity inhibitor is administeredintraperitoneally. Suitable STAT3 inhibitors include pyrimethamine,atovaquone, pimozide, guanabenz acetate, alprenolol hydrochloride,nifuroxazide, solanine alpha, fluoxetine hydrochloride, ifosfamide,pyrvinium pamoate, moricizine hydrochloride,3,3′-oxybis[tetrahydrothiophene, 1,1,1′,1′-tetraoxide],3-(1,3-benzodioxol-5-yl)-1,6-dimethyl-pyrimido[5,4-e]-1,2,4-triazine-5,7(-1H,6H)-dione, 2-(1,8-Naphthyridin-2-yl)phenol,3-(2-hydroxyphenyl)-3-phenyl-N,N-dipropylpropanamide as well as anyderivatives of these compounds or analogues thereof.

In one aspect, the STAT3 activity inhibitor comprises JSI-124(cucurbitacin I). For example, the JSI-124 (cucurbitacin I) isadministered at a dose of about 0.01 μM to about 10.0 μM, e.g., 0.01 μM,0.02 μM, 0.03 μM, 0.04 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM,0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM,2.0 μM, 3.0 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, or 10.0μM. Alternatively, the JSI-124 is administered at a dose of 1 mg/kg/day.

For example, the STAT3 inhibitor is administered three times per day,once per day, three times per week, once per week, three times permonth, once per month, once every three months, or once every sixmonths. In some cases, the STAT3 inhibitor is administered for onemonth, three months, six months, one year, or more.

The subject's gynecological cancer is inhibited, e.g., by at least 5%,at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or 100%. In oneexample, tumor cell growth in the subject's abdomen is inhibited. Forexample, the methods described herein prevent ovarian tumor cellspheroid formation, disrupt and disintegrate spheroids, preventattachment of spheroids to other organs, and prevent the development ofmetastases. In another example, subcutaneous tumor cell growth in thesubject is inhibited. In yet another aspect, ovarian tumor cellmetastases in the subject are inhibited. In some cases, malignantabdominal fluid (ascites) is inhibited, e.g., tumor spheroids inmalignant abdominal fluid are inhibited.

The methods described herein prevent tumor recurrence. For example,tumor recurrence is inhibited for one week, one month, three months, sixmonths, one year, three years, five years, or more.

In some cases, the methods further comprise administering atherapeutically effective amount of a chemotherapeutic agent. Forexample, the chemotherapeutic agent comprises a platinum-basedchemotherapeutic agent or a taxane-based chemotherapeutic agent.Suitable platinum-based chemotherapeutic agents include cisplatin andcarboplatin. For example, the chemotherapeutic agent is administeredprior to, simultaneously with, or subsequent to administration of theSTAT3 activity inhibitor.

In one aspect, the subject has received prior treatment for thegynecological tumor. In some cases, the gynecological tumor is resistantto platinum-based chemotherapy. For example, the subject has minimalresidual disease (MRD) following platinum-based chemotherapy.

Methods for treating a gynecological tumor in a subject are carried outby identifying a subject with a gynecological tumor, administering tothe subject a therapeutically effective amount of a chemotherapeuticagent to inhibit the gynecological tumor, and administering to thesubject a therapeutically effective amount of a STAT3 activity inhibitorto prevent recurrence of the gynecological tumor and development ofmetastases, thereby treating or preventing the gynecological tumor inthe subject. For example, the STAT3 activity inhibitor is administeredat least one month after administration of the chemotherapeutic agent,e.g., at least 2 months, at least 3 months, at least 4 months, at least5 months, at least 6 months, at least 9 months, or at least 1 year afteradministration of the chemotherapeutic agent.

Also provided are methods for treating a platinum-resistantgynecological tumor in a subject comprising identifying a subject with aplatinum-resistant gynecological tumor, administering to the subject atherapeutically effective amount of a STAT3 activity inhibitor, andadministering to the subject a therapeutically effective amount of achemotherapeutic agent, thereby treating the platinum-resistantgynecological tumor in the subject. In some cases, the STAT3 activityinhibitor is administered prior to administration of thechemotherapeutic agent, e.g., 1 month, 2 months, 3 months, 4 months, 5months, 6 months, 9 months, 1 year, or more prior to administration ofthe chemotherapeutic agent.

Also, described herein are methods of determining and/or monitoring agene expression profile in a subject with ovarian cancer. Specifically,these methods are carried out by identifying a subject with ovariancancer; providing a tumor sample from the subject; disaggregating thetumor sample into a population of single cells; performing single-cellRNA sequencing (scRNA-seq) on the sample, thereby determining a geneexpression profile in a subject with ovarian cancer. In some cases, thedisaggregation further comprises removing red blood cells from thetissue sample. In one aspect, the ovarian cancer is resistant totherapy. Optionally, the gene expression profile is determined prior toadministration of therapy to the subject. Alternatively, the geneexpression profile is determined at the time of minimal residual disease(MRD) or relapse. For example, the tissue sample is dissected intopieces <10 mm³, e.g., less than 9 mm³, less than 8 mm³, less than 7 mm³,less than 6 mm³, less than 5 mm³, less than 4 mm³, less than 3 mm³, lessthan 2 mm³, or less than 1 mm³. Preferably, the tissue sample isdissected into pieces <1 mm³, e.g., less than 0.1 mm³ or less than 0.01mm³. Optionally, the tissue sample is dissected with a scalpel.

Also provided are methods of treating or preventing ovarian cancercomprising administering 1 mg/kg/day of JSI-124 (cucurbitacin I) to thesubject. For example, JSI-124 is administered at a dose of 10 uM. Insome cases, monitoring of the ovarian cancer is performed prior to andafter administration of a therapeutic agent to determine a resistanceprofile of the therapeutic agent.

In another aspect, the present invention provides for a method oftreating platinum-based chemotherapy resistant ovarian cancer comprisingtreating a subject in need thereof with an inhibitor of the JAK/STATpathway. The subject may have minimal residue disease (MRD) or theovarian cancer is a relapse. The inhibitor may be JSI-124. The inhibitormay comprise a therapeutic antibody, bi-specific antibody, antibodyfragment, antibody-like protein scaffold, aptamer, genetic modifyingagent or small molecule.

In another aspect, the present invention provides for a method oftreating ovarian cancer comprising treating a subject in need thereofwith an agent capable of inhibiting or modulating expression or activityof one or more genes or polypeptides selected from the group consistingof JAK1, STAT3, STAT2, STAT1, OSMR, STAT6, RELA, ERBB2, GF1R, ERBB3,IL10RB, FGFR1, CXCR4, CXCL2, CXCL10, CXCL11, HLA-DRA, HLA-DRB1,HLA-DQB1, IDO1, MYBL2 and FGF13 or a gene selected from FIG. 4D,E. Incertain embodiments, the agent comprises a therapeutic antibody,bi-specific antibody, antibody fragment, antibody-like protein scaffold,aptamer, genetic modifying agent or small molecule. The gene orpolypeptide may be a surface or secreted gene. The agent may target asecreted protein or a receptor for the secreted protein. The agent maytarget a ligand for a surface receptor. The method may further compriseadministering platinum-based chemotherapy.

In certain embodiments, the ovarian cancer is chemotherapy resistant. Incertain embodiments, the ovarian cancer is platinum-based chemotherapyresistant.

In another aspect, the present invention provides for a method ofpredicting a response to platinum-based chemotherapy in a subjectsuffering from ovarian cancer comprising detecting in a tumor sampleobtained from the subject expression of one or more genes selected fromthe group consisting of JAK1, STAT3, STAT2, STAT1, OSMR, STAT6, RELA,ERBB2, GF1R, ERBB3, IL10RB, FGFR1, CXCR4, CXCL2, CXCL10, CXCL11,HLA-DRA, HLA-DRB1, HLA-DQB1, IDO1, MYBL2 and FGF13 or a gene selectedfrom FIG. 4D,E. In certain embodiments, high expression in the tumorsample indicates a weak response to platinum-based chemotherapy and lowexpression indicates a strong response to platinum-based chemotherapy.

In another aspect, the present invention provides for a method ofdetecting ovarian cancer stem cells in a tumor sample obtained from asubject suffering from ovarian cancer comprising detecting expression ofa gene signature comprising one or more genes selected from the groupconsisting of ALDH1A3, CD24, CD133, FN1, ACTA2, MYL9, GAS6, IGFBP5,FGFR1, CALD1, CFI, CRB2, PRDX4, IGFBP6 and RPS20.

In another aspect, the present invention provides for a method oftreating ovarian cancer comprising treating a subject in need thereofwith an agent capable of targeting ovarian cancer stem cellscharacterized by a gene signature comprising one or more genes selectedfrom the group consisting of ALDH1A3, CD24, CD133, FN1, ACTA2, MYL9,GAS6, IGFBP5, FGFR1, CALD1, CFI, CRB2, PRDX4, IGFBP6 and RPS20.

The agent may target a surface protein on the ovarian cancer stem cells.The agent may comprise a therapeutic antibody, bispecific antibody,antibody fragment, antibody-like protein scaffold, aptamer or CAR Tcell. The method may further comprise administering platinum-basedchemotherapy.

The method of treatment according to any embodiment herein may beadministered as an adjuvant or neoadjuvant therapy.

These and other aspects, objects, features, and advantages of theexample embodiments will become apparent to those having ordinary skillin the art upon consideration of the following detailed description ofillustrated example embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the features and advantages of the present inventionwill be obtained by reference to the following detailed description thatsets forth illustrative embodiments, in which the principles of theinvention may be utilized, and the accompanying drawings of which:

FIG. 1A-FIG. 1D are a series of dot plots, bar charts, and geneexpression profiles (heatmaps) depicting inter-tumor heterogeneity andcharting the ovarian cancer ascites by droplet-based single cellRNA-seq. Heterogeneity of malignant and non-malignant cells wasassessed. FIG. 1A is a t-distributed stochastic neighbor embedding(tSNE) analysis dot plot of number of single cells from number ofsamples profiled by the droplet-based scRNA-seq. Cells are colored bytheir assignment to 20 clusters whose indices are indicated in thecenter of each cluster. FIG. 1B is a bar plot showing the number ofcells from each sample which are assigned to each cluster. FIG. 1C is aheatmap of average expression in each of the clusters. Included are thetop 30 cluster-specific genes of each of the clusters, which arepresented in the order defined by hierarchical clustering. Subsets ofgenes which include cell type-specific markers are highlighted (lines tothe right of the heatmap). FIG. 1D is a heatmap of average expression ineach of the clusters that correspond to ovarian cancer cells (left),macrophages (middle) and fibroblasts (right). In each panel, includedare the top 30 cluster-specific genes of each cluster (compared to otherclusters in that panel), which are presented in the order defined byhierarchical clustering.

FIG. 2A-FIG. 2E are a series of dot plots, bar charts, and geneexpression profiles (heatmaps) depicting tumor cell subtypes andhighlighting diversity of cancer cell expression profiles as defined byplate-based single cell RNA-seq. FIG. 2A is a tSNE dot plot of thenumber of single cells from the number of samples profiled by theplate-based single cell RNA-seq. Cells are colored by their assignmentto the clusters whose indices are indicated in the center of eachcluster. FIG. 2B is a bar plot showing the number of cells from eachsample which are assigned to each cluster. FIG. 2C is a heatmap ofaverage expression in each of the clusters. Included are the top 30cluster-specific genes of each of the clusters, which are presented inthe order defined by hierarchical clustering. Subsets of genes whichinclude cell type-specific markers are highlighted (lines to the rightof the heatmap). FIG. 2C also shows a heatmap with columns representingthe average expression of cells from clusters in the tSNE after specificinterrogation using defined signatures from TCGA (differentiated,proliferative, etc. FIG. 2D is a graph of inference of chromosomalcopy-number variations (CNVs) from gene expression. For each cluster,the relative copy number in each chromosomal position was estimated bythe average expression of the 100 genes surrounding that position. Left:this approach predicted widespread amplifications and deletions in eachof the cancer cell clusters (clusters 1-6 corresponding to the cancercells from each of the patient samples, respectively) compared to thenon-cancer cells which were used as a reference. Right: when randomlyordering the genes across the genome and repeating the analysis, thesignal for CNV is eliminated, supporting the predicted CNVs. FIG. 2E isa graph of subtype scores for cancer cell clusters and a gene expressionprofile (heatmap). Each of the clusters was scored for the four CancerGenome Atlas (TCGA) subtypes, based on the average expression ofsubtype-specific genes.

FIG. 3A-FIG. 3F are a series of graphs depicting expression modules withintra-tumoral variability among ovarian cancer cells from patientascites samples. Distinct and shared cell state modules in platinumresistance were found. Significant heterogeneity of cell states andmodules within tumors was observed. Shared programs involve cell cycleand immune related pathways (e.g. janus kinase/signal transducer andactivator of transcription (JAK/STAT) signaling). FIG. 3A is a heatmapshowing relative expression of module-specific genes across GL-13 cellsfrom patients. FIG. 3B is a heatmap showing relative expression ofmodule-specific genes across GL-15 cells from patients (modules inindividual tumors). FIG. 3C is a heatmap showing relative expression ofmodule-specific genes across GL-17 cells from patients. In each case,expression modules were defined by non-negative matrix factorization(NMF) and are shown from top to bottom by the top 30 module-specificgenes for each module, and with annotation of selected genes; cells wereordered by hierarchical clustering. FIG. 3D is a heatmap showing theoverlap in the top genes between each pair of modules (across allpatients) as a measure of module similarity. Modules were ordered byhierarchical clustering, and their patient-of-origin is indicated at thetop. FIG. 3E is a graph of shared programs of selected cell cycle (top)and immune-related (bottom) genes among the top genes of modules (acrossall patients), with modules ordered as in FIG. 3D. Genes among the 30 ofa given module are indicated by black squares while those only among thetop 50 genes are indicated by gray squares. FIG. 3F is a graph ofexpression of highly-abundant transcripts in single-cells indicatedisproportionally high expression of key nodes of the JAK/STAT pathway,including JAK1, STAT1, STAT3, and others.

FIG. 4A-FIG. 4E are a series of graphs depicting comparisons ofexpression profiles across patient-derived xenograft (PDX) models beforeand after recurrence. Probing platinum-resistance in PDX-models revealsJAK/STAT signaling as shared resistance program. FIG. 4A is a plot oftumor burden of PDX model DF20. PDX model DF20 response to carboplatinwas assessed. Triangles indicate vehicle mice, squares indicate relapsedmice, and asterisks indicates MRD animals. MRD and relapsed tumors weretreated with three cycles of carboplatin and harvested for single-cellRNA-sequencing at the last indicated time point in this graph. FIG. 4Bis a heatmap showing the similarities and clustering of averageexpression profiles for each of the samples corresponding to three PDXmodels, with samples collected before treatments (vehicle), MRD and fullrelapse for each of the models. FIG. 4C is a plot of expression ofhighly-abundant transcripts in single-cells indicate disproportionallyhigh expression of key nodes of the JAK/STAT pathway, including STAT2and STAT3, and others. FIG. 4D is a heatmap showing differentialexpression between each MRD sample and the vehicle samples of therespective PDX model, for genes which are upregulated in at least twoMRD samples; genes were ordered by hierarchical clustering. FIG. 4E is aheatmap showing differential expression between each relapse sample andthe vehicle samples of the respective PDX model, for genes which areupregulated in at least two relapse samples; genes were ordered byhierarchical clustering.

FIG. 5A-FIG. 5E are a series of graphs depicting expression modules withintra-tumoral variability among ovarian cancer cells from PDX samples.FIG. 5A is a heatmap showing relative expression of module-specificgenes across DF20 cancer cells from PDX models. FIG. 5B is a heatmapshowing relative expression of module-specific genes across DF68 cancercells from PDX models. FIG. 5C is a heatmap showing relative expressionof module-specific genes across DF101 cancer cells from PDX models. Ineach case, expression modules were defined by NMF and are shown from topto bottom by the top 30 module-specific genes for each module, and withannotation of selected genes; cells were ordered by hierarchicalclustering. FIG. 5D is a heatmap showing the overlap in the top genesbetween each pair of modules from PDX samples (columns) and patientascites samples (rows) as a measure of module similarity. Patientascites modules were ordered as in FIG. 3D, and PDX modules were orderedby hierarchical clustering, with their model-of-origin indicated at thetop. FIG. 5E selected cell cycle (top), immune-related (middle) andother genes (bottom) among the top genes of each PDX module, withmodules ordered as in FIG. 5D. All genes included were shared betweenthe corresponding PDX module and the highest-overlapping patient ascitesmodules. Genes which are among the top 30 of a given module areindicated by black squares while those only among the top 50 genes areindicated by gray squares.

FIG. 6A-FIG. 6G are a series of graphs and images depicting the impactof JAK/STAT-inhibition on spheroid formation, invasion and killing exvivo and in vitro. FIG. 6A a histogram (FIG. 11B), showing drugscreening with 14 compounds (at 1 μM) inhibiting the JAK/STAT pathway inOVACR4. Relative viability compared to DMSO control measured by CTG.JSI-124 shows significant activity compared to other JAK/STATinhibitors. The same library was screened in 2D-cultures of OVACR4 (FIG.11A), and 2D-cultures of OVACR8 (FIG. 11C) and 3D-cultures of OVACR8(FIG. 11D). FIG. 6B is a series of plots showing drug sensitivitytesting using the GILA assay in platinum-resistant ex vivo spheroidcultures of patients DF3291 and DF3266. The X-axis indicates the log μMconcentration of drugs indicated in the legend, Y-axis indicates thepercentage of luminescence signal as indicator for viability relativelyto DMSO treated cells. In both patients, half maximal effectiveconcentration (EC₅₀) were ˜100 nM and ˜10 nM, respectively, while otherdrugs routinely used for the treatment of (platinum-resistant) ovariancancer had EC₅₀>10 μM. FIG. 6C is a series of images of microscopicanalysis of spheroids with different drug treatments to highlight themorphologic changes during therapy with drugs listed. Inhibition ofJAK/STAT pathway disrupts ex vivo spheroid cultures. FIG. 6D is ahistogram depicting impact of JSI-124 compared to carboplatin andcontrol on the formation of spheroids in five established ovarian cancercell lines. The X-axis indicates established ovarian cancer cell lineID. Y-axis indicates the relative number of formed spheroids whentreated with either JSI-124 or carboplatin compared to untreatedparental controls. Error bars indicate the standard deviation from themean number of spheroids. At 100 nM, JSI-124 completely abolished theability of cell lines to establish spheroids. JAK/STAT inhibitionprevents spheroid formation. FIG. 6E is a histogram depictingquantitative assessment of mesothelial clearance by patient-derivedspheroids (NCAT8) with prior treatment with JSI-124 (for either 30minutes or 120 minutes) vs. DMSO. The Y-axis indicates the clearanceability (using arbitrary units). 20 spheroids clusters were analyzed foreach iteration. Data is shown as mean (horizontal bar), interquartilerange (box), and minimum and maximum (whiskers). Statistical analysiswas performed using 1-way analysis of variance (ANOVA) and post hocTukey-Kramer **P<0.05. FIG. 6F is a histogram depicting resultsfollowing treatment with JSI-124 for 30 minutes of the two ovariancancer cell lines OVCAR8 and TYKNU. FIG. 6G is a histogram showing theinhibition of the JAK/STAT pathway.

FIG. 7A-FIG. 7E are a series of graphs and images that depict the impactof JAK/STAT-inhibition on spheroid formation in vivo and anti-tumoractivity. FIG. 7A is a diagram of a study design to investigate theeffect of JSI-124 on PDX model DF20. Each of these were performedfollowing intraperitoneal (IP) or subcutaneous (SC) injection of cancercells followed by IP therapy with JSI-124 vs. vehicle. Each groupincludes 5 mice. The inverted triangle indicates the time=0 of cancercell injection. FIG. 7B is a plot of a comparison of mice injected withDF20 intraperitoneally (IP) and treated with JSI-124 (IP) or vehicle.Mice were injected IP with tumor cells and one week later startedtreatment for a total 14 days. Each group includes 5 mice. Y-axisindicates log of bioluminescence imaging (BLI) signal (log total flux inp/s). Values are given as mean of log BLI signal, error bars indicatestandard deviation from the mean. ***p<0.0001 (two-sided t testcomparing mean±STDEV at Day 15 of treatment). FIG. 7C is a plot of acomparison of mice injected with DF20 IP and treated with JSI-124 orvehicle. Mice were injected IP with tumor cells and malignant asciteswas allowed to form for 21 days followed by treatment with JSI-124 (IP)or vehicle for a total 14 days. Each group includes 5 mice. Y-axisindicates log BLI signal (log total flux in p/s). Values are given asmean of log BLI signal, error bars indicate standard deviation from themean. ***p<0.0001 (two-sided t test comparing mean±STDEV at Day 15 oftreatment). FIG. 7D is a plot of a comparison of mice injected with DF20subcutaneously (SC) and treated with JSI-124 (IP) or vehicle. Mice wereinjected SC with tumor cells and one week later started treatment for atotal 14 days. Each group includes 5 mice. Y-axis indicates log BLIsignal (log total flux in p/s). Values are given as mean of log BLIsignal, error bars indicate standard deviation from the mean.***p<0.0001 (two-sided t test comparing mean±STDEV at Day 15 oftreatment). FIG. 7E is a plot of a comparison of mice injected with DF20SC and treated with JSI-124 or vehicle. Mice were injected SC with tumorcells and tumors were allowed to form for 21 days followed by treatmenttreatment with JSI-124 (IP) or vehicle for a total 14 days. Each groupincludes 5 mice. Y-axis indicates log BLI signal (log total flux inp/s). Values are given as mean of log BLI signal, error bars indicatestandard deviation from the mean. ***p<0.0001 (two-sided t testcomparing mean±STDEV at Day 15 of treatment).

FIG. 8A-FIG. 8E are a series of graphs depicting expression modules frompatient ascites samples and a putative stemness program. FIG. 8A is aheatmap showing relative expression of module-specific genes acrossGL10_cancer cells from patients. FIG. 8B is a heatmap showing relativeexpression of module-specific genes across GL17 cancer cells frompatients. In each case, expression modules were defined by NMF and areshown from top to bottom by the top 30 module-specific genes for eachmodule, and with annotation of selected genes; cells were ordered byhierarchical clustering. FIG. 8C is a heatmap showing the relativeexpression of three putative stemness markers in cells from GL10, sortedby their average expression of the three markers. FIG. 8D is a heatmapshowing the relative expression of the top genes positively (top) ornegatively (bottom) correlated with the stemness scores shown in FIG. 8Cin cells from GL10, as in FIG. 8C. FIG. 8E is the same heatmap as FIG.8D for GL13 cells, demonstrating that the expression program associatedwith stemness markers in GL10 is not conserved in other patient ascitessamples, with GL13 as an example. Shown are the genes positively (top)or negatively (bottom) correlated with the stemness scores of GL10cells, for GL13 cells sorted by their average expression of the putativestemness markers.

FIG. 9 is a graph of changes in highly-expressed genes in pre-treatment,MRD and relapse in PDX-models identifies high expression of JAK/STATpathway nodes. Expression of highly-abundant transcripts in single-cellsindicate high expression of key nodes of the JAK/STAT pathway, includingSTAT3 and others. Colored dots indicate the expression of this gene ineither vehicle (blue), MRD (pink) or relapsed (red) PDX models.

FIG. 10 is a graph of the correlation of module expression ofinflammatory pathways among patients and PDX models.

FIG. 11A-FIG. 11E are a series of histograms of OVACR4 and OVACR8ovarian cancer cell lines grown in 2D and 3D cultures and treated with14 JAK/STAT pathway inhibitors at 1 μM. Viability of the cells wasdetermined using the CellTiterGlo assay. JSI-124 was identified as mosteffective compound leading to cell killing. FIG. 11A is a histogram ofOVCAR4 cells in high attachment dishes (2D culture). FIG. 11B is ahistogram of OVCAR4 cells in ultra low attachment dishes (3D culture).FIG. 11C is a histogram of OVCAR8 cells in high attachment dishes. FIG.11D is a histogram of OVCAR8 cells in ultra low attachment dishes. FIG.11E is a histogram of STAT3-dependent luciferase activity in Heya8 cellsthat were pre-treated with JSI-124 vs. DMSO for 1 hour and followed bystimulation of oncostatin M (OSM). This histogram shows JSI-124 leads toa significant reduction in STAT-dependent expression, indicating itson-target activity.

FIG. 12A-FIG. 12B are a series of graphs depicting JSI-124 effectivelykills ovarian cancer cell lines grown as two-dimensional (2D) culturesor three-dimensional (3D) spheroids using ultra-low attachment growthconditions. Inhibition of JAK/STAT pathway kills 2D and 3D cultured celllines FIG. 12A is a series of plots of ovarian cancer cell lines weregrown for 4 days in ultra-low attachment conditions eliciting formationof spheroids, followed by treatment with JSI-124, carboplatin,paclitaxel, cisplatin or olaparib at indicated doses (log μMconcentrations as indicated on X-axis). Viability was measured asrelative luminescence signal compared to DMSO control. FIG. 12B is aseries of plots depicting ovarian cancer cell liens were grown for 4days in regular plastic culture surfaces and treated with drugs asindicated in FIG. 12A.

FIG. 13 is a schematic illustrating that 75% of ovarian cancer cancerare diagnosed at stage III/IV. Stage I >90%; stage II 70%; stage III40%; stage IV 70%.

FIG. 14 is a schematic illustrating the typical results of ideal therapyfor ovarian cancer.

FIG. 15 is a schematic illustrating ascites in the female reproductivesystem. Ascites—the chicken and the egg. Ascites is a multicellularecosystem including malignant cells, macrophages, and cancer-associatedfibroblasts. One third (⅓) of all patients present with ascites—almostall patients at recurrence (Shield K, Gynecology Oncol, 2009).

FIG. 16A-FIG. 16C are a series of bar charts and a gene expressionprofile illustrating genomic characterization of ovarian cancer (OvCa)and platinum-resistance. FIG. 16A is a graph depicting RDG Q value(TCGA, Nature 2012). FIG. 16B is a heatmap depicting tumor and geneexpression groups. FIG. 16C is histograms of the number of mutations andnumber of non-silent coding mutations in tumors and ascites (Patch,Nature 2015).

FIG. 17 is an illustration demonstrating the rationale of single-cellsequencing compared to bulk sequencing.

FIG. 18 is a schematic showing the design of a study using cancerpatient samples to complete single-cell profiling and drug testing ofascites. Both droplet based sing-cell RNA-seq and plate basedsingle-cell RNA-seq are used for ascites assessment.

FIG. 19A-FIG. 19B are a series of heatmaps depicting ovarian cancerheterogeneity. FIG. 19A is a heatmap assaying unique cell states of GL10cells. In this effort, isolated single-cells from malignant effusionsfrom patient with highly resistant ovarian cancer. One hundred and sixsingle-cell transcriptomes from a patient were analyzed and CNVs wereinferred. For ovarian cancer, a large burden of CNVs was found. Whilemost of the CNVs were shared among cells, subpopulations were identifiedthat carried unique aberrations, such as chromosome 12 deletion. FIG.19B is an additional heatmap depicting isolated single-cells frommalignant effusions.

FIG. 20A-FIG. 20B are graphs of cancer treatment resistance inPDX-models. FIG. 20A is a line graph depicting probingplatinum-resistance in ovarian PDX-models (Liu J et al, CcR 2016). FIG.20B is a series of photographs of mice showing results of probing cancertreatment resistance in PDX-models.

FIG. 21 is a series of line graphs showing the results of probingminimal residual disease (MRD) and relapse at single-cell resolutionfrom DF-20 PDX model.

FIG. 22 is a diagram depicting immune cells and JAK/STAT expression inplatinum-resistant ovarian cancer cells. A common feature of resistantcells in patient ascites and in PDX models is the cancer cell-autonomousexpression on inflammatory pathways, including the JAK/STAT pathway.

FIG. 23 is an image of cancer cells from video 1 and 2 (top row) andvideo 3 and 4 (bottom row) exposed to DMSO, STAT3 inhibitor for 30 minand STAT3 inhibitor for 120 min. JAK/STAT inhibition prevents spheroidformation. Further description of video 1, 2, 3, and 4 is given in theexamples sections below.

FIG. 24 is a histogram of relative mRNA expression depicting STAT targetgene expression.

FIG. 25 is a schematic for treatment according to the JSI-124(cucurbitacin I) prevention therapy arm.

FIG. 26 is a schematic for treatment according to the JSI-124established tumor therapy arm.

FIG. 27 is a schematic showing the effect of intraperitoneal, adjuvantJAK/STAT3 inhibition on growth of SC tumors (DF-20). Endpoint: Time totumor recurrence.

FIG. 28 is a schematic showing the effect of IV, adjuvant JAK/STAT3inhibition on growth of SC tumors (DF-20). Endpoint: Time to tumorrecurrence.

FIG. 29 is a photograph, wherein the right area contained 10 μM JSI124+blue dye, left area served as a control (patient NACT14).

FIG. 30 is a series of photomicrographs showing the area with 10 μMJSI124 (patient NACT14 at day 1).

FIG. 31 is a series of photomicrographs showing the area without JSI124(patient NACT14 at day 1).

FIG. 32 is a series of photomicrographs showing 48 hours with 10 μMJSI124 (patient NACT14).

FIG. 33 is a series of photomicrographs showing 48 hours with 10 μMJSI124 (patient NACT14).

FIG. 34 is a series of photomicrographs showing 48 hours with 10 μMJSI124 (patient NACT14).

FIG. 35 is a series of photomicrographs showing 48 hours with 10 μMJSI124 (patient NACT14).

FIG. 36 is a series of photomicrographs showing morphologicaldescription of ex-vivo patient-derived ovarian cells after treatmentwith STAT3 inhibitor JSI124. JSI124-treated cells were observed with amajority of intact immune cells, due to massive elimination of tumorcells.

FIG. 37 is a dot plot showing mesothelial clearance.

FIG. 38 is a diagram of intraperitoneal therapy for cancer.

FIG. 39 is a graph comparing intraperitoneal therapy and intravenous(IV) therapy (Armstrong, NEJM, 2006).

FIG. 40 is a diagram of an in-vivo study design evaluating tumorformation in mice to evaluate activity of intraperitoneal administeredJAK/STAT inhibitor.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS General Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Definitions of common termsand techniques in molecular biology may be found in Molecular Cloning: ALaboratory Manual, 2^(nd) edition (1989) (Sambrook, Fritsch, andManiatis); Molecular Cloning: A Laboratory Manual, 4^(th) edition (2012)(Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (AcademicPress, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B.D. Hames, and G. R. Taylor eds.): Antibodies, A Laboraotry Manual (1988)(Harlow and Lane, eds.): Antibodies A Laboraotry Manual, 2^(nd) edition2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney,ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008(ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829);Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology andMolecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed.,John Wiley & Sons (New York, N.Y. 1992); “Oligonucleotide Synthesis”(Gait, 1984); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991); and Marten H. Hofkerand Jan van Deursen, Transgenic Mouse Methods and Protocols, 2^(nd)edition (2011).

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The term “optional” or “optionally” means that the subsequent describedevent, circumstance or substituent may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The terms “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, are meant to encompass variations of and from thespecified value, such as variations of +/−10% or less, +/−5% or less,+/−1% or less, and +1-0.1% or less of and from the specified value,insofar such variations are appropriate to perform in the disclosedinvention. It is to be understood that the value to which the modifier“about” or “approximately” refers is itself also specifically, andpreferably, disclosed. Unless specifically stated or obvious fromcontext, as used herein, the term “about” is understood as within arange of normal tolerance in the art, for example within 2 standarddeviations of the mean. “About” can be understood as within 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the statedvalue. Unless otherwise clear from context, all numerical valuesprovided herein are modified by the term “about.”

As used herein, a “biological sample” may contain whole cells and/orlive cells and/or cell debris. The biological sample may contain (or bederived from) a “bodily fluid”. The present invention encompassesembodiments wherein the bodily fluid is selected from amniotic fluid,aqueous humour, vitreous humour, bile, blood serum, breast milk,cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph,perilymph, exudates, feces, female ejaculate, gastric acid, gastricjuice, lymph, mucus (including nasal drainage and phlegm), pericardialfluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skinoil), semen, sputum, synovial fluid, sweat, tears, urine, vaginalsecretion, vomit and mixtures of one or more thereof. Biological samplesinclude cell cultures, bodily fluids, cell cultures from bodily fluids.Bodily fluids may be obtained from a mammal organism, for example bypuncture, or other collecting or sampling procedures.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human. Inhibition of metastasis is frequently aproperty of antineoplastic agents.

By “agent” is meant any small compound, antibody, nucleic acid molecule,or polypeptide, or fragments thereof.

By “alteration” is meant a change (increase or decrease) in theexpression levels or activity of a gene or polypeptide as detected bystandard art-known methods such as those described herein. As usedherein, an alteration includes at least a 1% change in expressionlevels, e.g., at least a 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100% change in expression levels. Forexample, an alteration includes at least a 5%-10% change in expressionlevels, preferably a 25% change, more preferably a 40% change, and mostpreferably a 50% or greater change in expression levels.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity. The term “immunoglobulin” (Ig) is usedinterchangeably with “antibody” herein.

By “binding to” a molecule is meant having a physicochemical affinityfor that molecule. By “control” or “reference” is meant a standard ofcomparison. As used herein, “changed as compared to a control” sample orsubject is understood as having a level that is statistically differentthan a sample from a normal, untreated, or control sample. Controlsamples include, for example, cells in culture, one or more laboratorytest animals, or one or more human subjects. Methods to select and testcontrol samples are within the ability of those in the art. An analytecan be a naturally occurring substance that is characteristicallyexpressed or produced by the cell or organism (e.g., an antibody, aprotein) or a substance produced by a reporter construct (e.g.,β-galactosidase or luciferase). Depending on the method used fordetection, the amount and measurement of the change can vary.Determination of statistical significance is within the ability of thoseskilled in the art, e.g., the number of standard deviations from themean that constitute a positive result.

“Detect” refers to identifying the presence, absence, or amount of theagent (e.g., a nucleic acid molecule, for example deoxyribonucleic acid(DNA) or ribonucleic acid (RNA)) to be detected.

By “detectable label” is meant a composition that when linked (e.g.,joined—directly or indirectly) to a molecule of interest renders thelatter detectable, via, for example, spectroscopic, photochemical,biochemical, immunochemical, or chemical means. Direct labeling canoccur through bonds or interactions that link the label to the molecule,and indirect labeling can occur through the use of a linker or bridgingmoiety which is either directly or indirectly labeled. Bridging moietiesamplifies a detectable signal. For example, useful labels includeradioactive isotopes, magnetic beads, metallic beads, colloidalparticles, fluorescent labeling compounds, electron-dense reagents,enzymes (for example, as commonly used in an enzyme-linked immunosorbentassay (ELISA)), biotin, digoxigenin, or haptens. When the fluorescentlylabeled molecule is exposed to light of the proper wave length, itspresence can then be detected due to fluorescence. Among the mostcommonly used fluorescent labeling compounds are fluoresceinisothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,p-phthaldehyde and fluorescamine. The molecule can also be detectablylabeled using fluorescence emitting metals such as 152 Eu, or others ofthe lanthanide series. These metals can be attached to the moleculeusing such metal chelating groups as diethylenetriaminepentacetic acid(DTPA) or ethylenediaminetetraacetic acid (EDTA). The molecule also canbe detectably labeled by coupling it to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged molecule is then determined bydetecting the presence of luminescence that arises during the course ofchemical reaction. Examples of particularly useful chemiluminescentlabeling compounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

A “detection step” may use any of a variety of known methods to detectthe presence of nucleic acid. The types of detection methods in whichprobes can be used include Western blots, Southern blots, dot or slotblots, and Northern blots.

As used herein, the term “diagnosing” refers to classifying pathology ora symptom, determining a severity of the pathology (e.g., grade orstage), monitoring pathology progression, forecasting an outcome ofpathology, and/or determining prospects of recovery.

By the term “disaggregate” is meant to separate something into itscomponent parts. Thus, “disaggregating” a tissue sample into apopulation of single cells means to separate a tissue sample into thesingle cells which together form the tissue sample.

By the terms “effective amount” and “therapeutically effective amount”of a formulation or formulation component is meant a sufficient amountof the formulation or component, alone or in a combination, to providethe desired effect. For example, by “an effective amount” is meant anamount of a compound, alone or in a combination, required to amelioratethe symptoms of a disease, e.g., cancer, relative to an untreatedpatient. The effective amount of active compound(s) used to practice thepresent invention for therapeutic treatment of a disease variesdepending upon the manner of administration, the age, body weight, andgeneral health of the subject. Ultimately, the attending physician orveterinarian will decide the appropriate amount and dosage regimen. Suchamount is referred to as an “effective” amount.

The term “expression profile” is used broadly to include a genomicexpression profile. Profiles may be generated by any convenient meansfor determining a level of a nucleic acid sequence, e.g., quantitativehybridization of microRNA, labeled microRNA, amplified microRNA,complementary/synthetic DNA (cDNA), etc., quantitative polymerase chainreaction (PCR), and ELISA for quantitation, and allow the analysis ofdifferential gene expression between two samples. A subject or patienttumor sample is assayed. Samples are collected by any convenient method,as known in the art. According to some embodiments, the term “expressionprofile” means measuring the relative abundance of the nucleic acidsequences in the measured samples.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. For example, a fragment maycontain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 nucleotides or amino acids. However, theinvention also comprises polypeptides and nucleic acid fragments, solong as they exhibit the desired biological activity of the full lengthpolypeptides and nucleic acid, respectively. A nucleic acid fragment ofalmost any length is employed. For example, illustrative polynucleotidesegments with total lengths of about 10,000, about 5000, about 3000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length (including all intermediate lengths) are included inmany implementations of this invention. Similarly, a polypeptidefragment of almost any length is employed. For example, illustrativepolypeptide segments with total lengths of about 10,000, about 5,000,about 3,000, about 2,000, about 1,000, about 5,000, about 1,000, about500, about 200, about 100, or about 50 amino acids in length (includingall intermediate lengths) are included in many implementations of thisinvention.

“Hybridization” means hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementarynucleobases. For example, adenine and thymine are complementarynucleobases that pair through the formation of hydrogen bonds.

By “hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation.

A “purified” or “biologically pure” protein is sufficiently free ofother materials such that any impurities do not materially affect thebiological properties of the protein or cause other adverseconsequences. That is, a nucleic acid or peptide of this invention ispurified if it is substantially free of cellular material, viralmaterial, or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.Purity and homogeneity are typically determined using analyticalchemistry techniques, for example, polyacrylamide gel electrophoresis orhigh performance liquid chromatography. The term “purified” can denotethat a nucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. For a protein that can be subjected tomodifications, for example, phosphorylation or glycosylation, differentmodifications may give rise to different isolated proteins, which can beseparately purified.

Similarly, by “substantially pure” is meant a nucleotide or polypeptidethat has been separated from the components that naturally accompany it.Typically, the nucleotides and polypeptides are substantially pure whenthey are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, freefrom the proteins and naturally-occurring organic molecules with theyare naturally associated.

By “isolated nucleic acid” is meant a nucleic acid that is free of thegenes which flank it in the naturally-occurring genome of the organismfrom which the nucleic acid is derived. The term covers, for example:(a) a DNA which is part of a naturally occurring genomic DNA molecule,but is not flanked by both of the nucleic acid sequences that flank thatpart of the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner, such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a synthetic cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein. Isolatednucleic acid molecules according to the present invention furtherinclude molecules produced synthetically, as well as any nucleic acidsthat have been altered chemically and/or that have modified backbones.For example, the isolated nucleic acid is a purified cDNA or RNApolynucleotide. Isolated nucleic acid molecules also include messengerribonucleic acid (mRNA) molecules.

By an “isolated polypeptide” is meant a polypeptide of the inventionthat has been separated from components that naturally accompany it.Typically, the polypeptide is isolated when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, a polypeptide of the invention. An isolated polypeptideof the invention may be obtained, for example, by extraction from anatural source, by expression of a recombinant nucleic acid encodingsuch a polypeptide; or by chemically synthesizing the protein. Puritycan be measured by any appropriate method, for example, columnchromatography, polyacrylamide gel electrophoresis, or byhigh-performance liquid chromatography (HPLC) analysis.

The term “immobilized” or “attached” refers to a probe (e.g., nucleicacid or protein) and a solid support in which the binding between theprobe and the solid support is sufficient to be stable under conditionsof binding, washing, analysis, and removal. The binding may be covalentor non-covalent. Covalent bonds may be formed directly between the probeand the solid support or may be formed by a cross linker or by inclusionof a specific reactive group on either the solid support or the probe orboth molecules. Non-covalent binding may be one or more ofelectrostatic, hydrophilic, and hydrophobic interactions. Included innon-covalent binding is the covalent attachment of a molecule to thesupport and the non-covalent binding of a biotinylated probe to themolecule. Immobilization may also involve a combination of covalent andnon-covalent interactions.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder, e.g., cancer.

By “modulate” is meant alter (increase or decrease). Such alterationsare detected by standard art-known methods such as those describedherein.

Relative to a control level, the level that is determined may be anincreased level. As used herein, the term “increased” with respect tolevel (e.g., expression level, biological activity level, etc.) refersto any % increase above a control level. The increased level may be atleast or about a 1% increase, at least or about a 5% increase, at leastor about a 10% increase, at least or about a 15% increase, at least orabout a 20% increase, at least or about a 25% increase, at least orabout a 30% increase, at least or about a 35% increase, at least orabout a 40% increase, at least or about a 45% increase, at least orabout a 50% increase, at least or about a 55% increase, at least orabout a 60% increase, at least or about a 65% increase, at least orabout a 70% increase, at least or about a 75% increase, at least orabout a 80% increase, at least or about a 85% increase, at least orabout a 90% increase, or at least or about a 95% increase, relative to acontrol level.

Relative to a control level, the level that is determined may be adecreased level. As used herein, the term “decreased” with respect tolevel (e.g., expression level, biological activity level, etc.) refersto any % decrease below a control level. The decreased level may be atleast or about a 1% decrease, at least or about a 5% decrease, at leastor about a 10% decrease, at least or about a 15% decrease, at least orabout a 20% decrease, at least or about a 25% decrease, at least orabout a 30% decrease, at least or about a 35% decrease, at least orabout a 40% decrease, at least or about a 45% decrease, at least orabout a 50% decrease, at least or about a 55% decrease, at least orabout a 60% decrease, at least or about a 65% decrease, at least orabout a 70% decrease, at least or about a 75% decrease, at least orabout a 80% decrease, at least or about a 85% decrease, at least orabout a 90% decrease, or at least or about a 95% decrease, relative to acontrol level.

Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule.

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

By “neoplasia” is meant a disease or disorder characterized by excessproliferation or reduced apoptosis. Illustrative neoplasms for which theinvention can be used include, but are not limited to pancreatic cancer,leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, acute myeloblastic leukemia, acute promyelocyticleukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, glioblastomamultiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma,schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

As used herein, “obtaining” as in “obtaining an agent” includessynthesizing, purchasing, or otherwise acquiring the agent.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

The phrase “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administering compounds of the present invention tomammals. The carriers include liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agent from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations.

By “protein” or “polypeptide” or “peptide” is meant any chain of morethan two natural or unnatural amino acids, regardless ofpost-translational modification (e.g., glycosylation orphosphorylation), constituting all or part of a naturally-occurring ornon-naturally occurring polypeptide or peptide, as is described herein.

The terms “preventing” and “prevention” refer to the administration ofan agent or composition to a clinically asymptomatic individual who isat risk of developing, susceptible, or predisposed to a particularadverse condition, disorder, or disease, and thus relates to theprevention of the occurrence of symptoms and/or their underlying cause.

The term “prognosis,” “staging,” and “determination of aggressiveness”are defined herein as the prediction of the degree of severity of theneoplasia, e.g., cancer, and of its evolution as well as the prospect ofrecovery as anticipated from usual course of the disease.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it is understood thatthe particular value forms another aspect. It is further understood thatthe endpoints of each of the ranges are significant both in relation tothe other endpoint, and independently of the other endpoint. It is alsounderstood that there are a number of values disclosed herein, and thateach value is also herein disclosed as “about” that particular value inaddition to the value itself. It is also understood that throughout theapplication, data are provided in a number of different formats and thatthis data represent endpoints and starting points and ranges for anycombination of the data points. For example, if a particular data point“10” and a particular data point “15” are disclosed, it is understoodthat greater than, greater than or equal to, less than, less than orequal to, and equal to 10 and 15 are considered disclosed as well asbetween 10 and 15. It is also understood that each unit between twoparticular units are also disclosed. For example, if 10 and 15 aredisclosed, then 11, 12, 13, and 14 are also disclosed.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 aswell as all intervening decimal values between the aforementionedintegers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,and 1.9. With respect to sub-ranges, “nested sub-ranges” that extendfrom either end point of the range are specifically contemplated. Forexample, a nested sub-range of an exemplary range of 1 to 50 maycomprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

The term “sample” as used herein refers to a biological sample obtainedfor the purpose of evaluation in vitro. Exemplary tissue samples for themethods described herein include tissue samples from tumors or thesurrounding microenvironment (i.e., the stroma). With regard to themethods disclosed herein, the sample or patient sample preferably maycomprise any body fluid or tissue. In some embodiments, the bodily fluidincludes, but is not limited to, blood, plasma, serum, lymph, breastmilk, saliva, mucous, semen, vaginal secretions, cellular extracts,inflammatory fluids, cerebrospinal fluid, feces, vitreous humor, orurine obtained from the subject. In some aspects, the sample is acomposite panel of at least two of a blood sample, a plasma sample, aserum sample, and a urine sample. In exemplary aspects, the samplecomprises blood or a fraction thereof (e.g., plasma, serum, fractionobtained via leukopheresis). Other samples include whole blood, serum,plasma, or urine. A sample can also be a partially purified fraction ofa tissue or bodily fluid.

A reference sample can be a “normal” sample, from a donor not having thedisease or condition fluid, or from a normal tissue in a subject havingthe disease or condition. A reference sample can also be from anuntreated donor or cell culture not treated with an active agent (e.g.,no treatment or administration of vehicle only). A reference sample canalso be taken at a “zero time point” prior to contacting the cell orsubject with the agent or therapeutic intervention to be tested or atthe start of a prospective study.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

A subject “suffering from or suspected of suffering from” a specificdisease, condition, or syndrome has a sufficient number of risk factorsor presents with a sufficient number or combination of signs or symptomsof the disease, condition, or syndrome such that a competent individualwould diagnose or suspect that the subject was suffering from thedisease, condition, or syndrome. Methods for identification of subjectssuffering from or suspected of suffering from conditions associated withcancer (e.g., cancer) is within the ability of those in the art.Subjects suffering from, and suspected of suffering from, a specificdisease, condition, or syndrome are not necessarily two distinct groups.

As used herein, “susceptible to” or “prone to” or “predisposed to” or“at risk of developing” a specific disease or condition refers to anindividual who based on genetic, environmental, health, and/or otherrisk factors is more likely to develop a disease or condition than thegeneral population. An increase in likelihood of developing a diseasemay be an increase of about 10%, 20%, 50%, 100%, 150%, 200%, or more.

The terms “treating” and “treatment” as used herein refer to theadministration of an agent or formulation to a clinically symptomaticindividual afflicted with an adverse condition, disorder, or disease, soas to effect a reduction in severity and/or frequency of symptoms,eliminate the symptoms and/or their underlying cause, and/or facilitateimprovement or remediation of damage. It will be appreciated that,although not precluded, treating a disorder or condition does notrequire that the disorder, condition or symptoms associated therewith becompletely eliminated.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

Various embodiments are described hereinafter. It should be noted thatthe specific embodiments are not intended as an exhaustive descriptionor as a limitation to the broader aspects discussed herein. One aspectdescribed in conjunction with a particular embodiment is not necessarilylimited to that embodiment and can be practiced with any otherembodiment(s). Reference throughout this specification to “oneembodiment”, “an embodiment,” “an example embodiment,” means that aparticular feature, structure or characteristic described in connectionwith the embodiment is included in at least one embodiment of thepresent invention. Thus, appearances of the phrases “in one embodiment,”“in an embodiment,” or “an example embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment, but may. Furthermore, the particular features,structures or characteristics may be combined in any suitable manner, aswould be apparent to a person skilled in the art from this disclosure,in one or more embodiments. Furthermore, while some embodimentsdescribed herein include some but not other features included in otherembodiments, combinations of features of different embodiments are meantto be within the scope of the invention. For example, in the appendedclaims, any of the claimed embodiments can be used in any combination.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, published patentdocuments, and patent applications cited herein are hereby incorporatedby reference to the same extent as though each individual publication,published patent document, or patent application was specifically andindividually indicated as being incorporated by reference. Genbank andNCBI submissions indicated by accession number cited herein areincorporated herein by reference.

Overview

Embodiments disclosed herein describe the examination of drugsensitivity of patient-derived cells isolated from ovarian cancerpatients' ascites. The initial observation was that cells obtained fromplatinum-resistant patient demonstrate resistance to platinum-basedchemotherapy also at the ex-vivo experimental system. Next, asingle-cell RNA-sequences from patient and animal models hinted that animmune gene signature/STAT3 pathway are involved in the recurrent of thedisease. A STAT3 inhibitor (JSI-124, Sigma-Aldrich®) was added to thestudy and experimental results indicated that the cells are verysensitive to the drug, as compared to the clinically-availablechemotherapy. Additionally, a functional assay supported experimentalresults by showing that the use of JSI-124 inhibits invasiveness ofspheres that originated from patient-derived cells. Inhibition ofinvasiveness was surprisingly fast and suggested a potential rationalefor the fast effect of JSI-124. Described herein is the treatment ofanimals by intra-peritoneum injection of JSI-124 and to test the effecton recurrent of tumor growth. The results presented herein indicate thatJSI-124 reduces malignancy, and in particular invasiveness, of tumorcells.

As described herein, targeting STAT3 helps to inhibit invasiveness oftumor cells (short term) and, together with chemotherapy, to prevent thespread of the disease (long term).

Prior to the invention described herein, injection of a STAT3 inhibitorinto the peritoneum was not examined for reduction of malignancy ofgynecological tumors. Additionally, prior to the invention describedherein, many treatments failed to prevent tumor recurrent, as usuallyhappens in ovarian cancer. Presented herein is an approach that directlyregulates the spread of the disease using a drug that demonstratessignificant efficiency as compared to the clinical drugs.

Tumors, including ovarian tumors, are complex ecosystems defined byspatiotemporal interactions between heterogeneous cell types, includingmalignant, immune, and stromal cells (D. Hanahan and R. A. Weinberg,2011 Cell, 144: 646-674). Each tumor's cellular composition, as well asthe interplay between these components, exerts critical roles in cancerdevelopment (C. E. Meacham and S. J. Morrison, 2013 Nature, 501:328-337). However, prior to the invention described herein, the specificcomponents, their salient biological functions, and the means by whichthey collectively define tumor behavior were incompletely characterizedin ovarian cancer.

Tumor cellular diversity poses both challenges and opportunities forcancer therapy. This is most clearly demonstrated by the remarkable, butvaried, clinical efficacy achieved in malignant melanoma with targetedtherapies and immunotherapies. First, immune checkpoint inhibitorsproduce substantial clinical responses in some patients with metastaticmelanomas (Hodi et al., 2010 N. Engl. J. Med., 363: 711-723; Brahmer etal., 2010 J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol., 28: 3167-3175;Brahmer et al., 2012 N. Engl. J. Med., 366: 2455-2465; Topalian et al.,2012 N. Engl. J. Med., 366: 2443-2454; and Hamid et al., 2013 N. Engl.J. Med., 369: 134-144); however, prior to the invention describedherein, the genomic and molecular determinants of response to theseagents was poorly understood. Collectively, these factors highlight theneed for a deeper understanding of melanoma composition and its impacton clinical course.

The next wave of therapeutic advances in cancer are accelerated byemerging technologies that systematically assess the malignant,microenvironmental, and immunologic states most likely to informtreatment response and resistance. An ideal approach would assesssalient cellular heterogeneity by quantifying variation in oncogenicsignaling pathways, drug-resistant tumor cell subsets, and the spectrumof immune, stromal and other cell states that inform immunotherapyresponse. Toward this end, emerging single-cell genomic approachesenable detailed evaluation of genetic and transcriptional featurespresent in 100s-1000s of individual cells per tumor (Shalek et al., 2013Nature, 498: 236-240; Patel et al., 2014 Science, 344: 1396-1401;Macosko et al., 2015 Cell, 161: 1202-1214). In principle, this approachprovides a comprehensive means to identify all major cellular componentssimultaneously and to determine their individual genomic and molecularstates (Patel et al., 2014 Science, 344: 1396-1401), and ascertain whichof these features predict or explain clinical responses to anticanceragents.

Resistance to therapy is a major impediment to improving outcomes inovarian cancer. Due to the lack of effective screening approaches, mostpatients are diagnosed at an advanced stage (Matulonis et al., 2016 Nat.Rev. Dis. Primer, 2:16061). Advanced-stage ovarian cancer is treatedwith surgery and chemotherapy with platinum and taxane agents. While10-15% of patients exhibit intrinsic resistance to initial chemotherapy,most patients typically have a good response and achieve diseaseremission following chemotherapy; however, residual disease isfrequently present and leads to relapse in 80% of patients within monthsto several years (Matulonis et al., 2016 Nat. Rev. Dis. Primer, 2:16061;Siegel et al., 2016 Cancer J. Clin., 66:7-30). Despite recent advancesin treatment, recurrent ovarian cancer is incurable and portends a poorprognosis with median survival of approximately one year (Siegel et al.,2016 Cancer J. Clin., 66:7-30). Development of resistance toplatinum-based chemotherapy and associated development malignantabdominal fluid (ascites) are the major impediment to improving outcomesfor patients with advanced ovarian cancer. To date, the mechanisms ofdrug resistance remain poorly understood. Lack of discovery of recurrentgenomic features of drug resistance is likely, in part, due to thesignificant heterogeneity of ovarian cancer, which biases analyses usingbulk tissue specimens or ascites fluid. Inter-tumor and particularlyintra-tumor heterogeneity of ovarian cancer cells and associatednon-cancer cells is an important factor driving treatment resistance,but remains poorly understood. New insights into molecular mechanisms ofintrinsic and acquired treatment resistance are required to identifycritical resistance pathways that reveal new therapeutic targets forrecurrent disease. Understanding inter-tumor and intra-tumorheterogeneity of ovarian cancer cells is a component of identifyingcritical resistance pathways and new therapeutic targets for recurrentdisease.

Inter-tumor and particularly intra-tumor heterogeneity of ovarian cancercells and associated non-cancer cells is an important factor drivingtreatment resistance, but remains poorly understood. High-grade serousovarian cancer (HGSC), the most common and aggressive histologicalsubtype, has been studied extensively by large-scale genomic studiessuch as TCGA, which revealed TP53 mutations, defects in homologousrecombination and extensive copy-number aberrations in most tumors(T.C.G.A.R. Network, 2011 Nature, 474:609-615). In addition to theseconsistent features, HGSC tumors were also classified intotranscriptional subtypes by TCGA and other studies of DNA repair andchemotherapy resistance (Patch et al., 2015 Nature, 521:489-494).However, these signatures have poor reproducibility (Lloyd et al., 2015BMC Cancer, 15:117) and are based on bulk profiles, which represent theaverage of cancer and non-cancer cells mixed within any tumor sample,and do not recapitulate the inherent tumor heterogeneity.

As described herein, single-cell RNA-sequencing offers a powerfulapproach for characterizing tumors by resolving the expression profilesof diverse cancer cells, infiltrating immune cells, and stromal cells,each of which contribute to treatment resistance through distinctmechanisms (Tirosh et al., 2016 Science, 352:189-196). In particular,bidirectional signaling between tumor cells and immune cells has beensuggested as a mode of ovarian cancer cell survival via immune evasion,and a deeper understanding of these pathways is critical to successfulapplication of immunotherapies (Gaillard et al., 2016 Gynecology Oncol.Res. Pract., 3:11). In addition to profiling of tumors with abundantcancer cells, the high sensitivity of scRNA-seq enables analysis ofsamples with low purity, such as ascites, or with very low cell numbers,such as minimal residual disease (MRD), which are not detectable usingcurrent methods. As described in detail below, scRNA-seq was applied toboth of these clinically important scenarios.

Ascites cancer cells, free-floating single cells and spheroids within apatient's peritoneal fluid, represent a critical feature of ovariancancer biology and clinical evolution. Ascites is present in one-thirdof patients at the time of diagnosis, and develops in the majority ofpatients with chemotherapy resistant disease (Ahmed et al., 2013 Front.Oncol., 3). Rather than a homogenous suspension of single cells, ascitesfluid is comprised of a multicellular collection of cancer cells, immunecells, and fibroblasts which contribute to disease progression (Kipps etal., 2013 Nat. Rev. Cancer, 13:273-282). Ovarian cancer spheroids withinascites are multicellular aggregates that can promote intraperitonealmetastasis and that are associated with chemotherapy resistance (Shieldet al., 2009 Gynecology Oncol., 113:143-148).

Presented herein is the first comprehensive analysis of patient ovariancancer ascites using scRNA-seq. Extensive heterogeneity of cancer cellsand cancer-associated macrophages and fibroblasts is demonstrated. Asdescribed in detail below, to examine the changes in tumor cellsoccurring during therapy and following relapse, scRNA-seq was performedon cells isolated from patient-derived xenograft (PDX) ovarian cancermodels pre-treatment, at minimal residual disease (MRD) and duringprogressive disease. As described herein, a common theme ofcancer-intrinsic inflammatory signaling, including the activation of theJAK/STAT pathway, was identified. As described in the examples blow,inhibition of the JAK2/STAT3 pathway is toxic to platinum-resistant,patient-derived ex vivo models, inhibits the formation, coherence andinvasive behavior of spheroids in vitro, and shows anti-tumor activityin PDX models, suggesting that JAK/STAT inhibition represents aclinically feasible therapeutic avenue for platinum-resistant ovariancancer.

Ovarian Cancer

Ovarian cancer is a cancer that forms in or on an ovary. Symptoms mayinclude bloating, pelvic pain, abdominal swelling, and loss of appetite,among others. Common areas to which the cancer may spread include thelining of the abdomen, lymph nodes, lungs, and liver. The most commontype of ovarian cancer is ovarian carcinoma (>95% of all cases). Thereare five main subtypes of ovarian carcinoma, of which high-grade serouscarcinoma is the most common. These tumors are believed to start in thecells covering the ovaries, though some may form at the Fallopian tubes.Less common types of ovarian cancer include germ cell tumors and sexcord stromal tumors. A diagnosis of ovarian cancer is confirmed througha biopsy of tissue, usually removed during surgery.

If caught and treated in an early stage, ovarian cancer is oftencurable. Treatment usually includes some combination of surgery,radiation therapy, and chemotherapy. Outcomes depend on the extent ofthe disease, the subtype of cancer present, and other medicalconditions. The overall five-year survival rate in the United States is45%.

If ovarian cancer recurs, it is considered partially platinum-sensitiveor platinum-resistant, based on the time since the last recurrencetreated with platins: partially platinum-sensitive cancers recurred 6-12months after last treatment, and platinum-resistant cancers have aninterval of less than 6 months.

For platinum-sensitive tumors, platins are utilized for second-linechemotherapy, often in combination with other cytotoxic agents. Regimensinclude carboplatin combined with pegylated liposomal doxorubicin,gemcitabine, or paclitaxel. If the tumor is determined to beplatinum-resistant, vincristine, dactinomycin, and cyclophosphamide(VAC) or some combination of paclitaxel, gemcitabine, and oxaliplatincan be used as a second-line therapy.

Prior to the invention described herein, there were no high-efficacychemotherapy options for platinum-resistant tumors.

STAT Molecules

Members of the signal transducer and activator of transcription (STAT)protein family are intracellular transcription factors that mediate manyaspects of cellular immunity, proliferation, apoptosis anddifferentiation. There are seven mammalian STAT family members that havebeen identified: STAT1, STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B),and STAT6. STAT proteins are primarily activated by membranereceptor-associated Janus kinases (JAK). Dysregulation of the JAK/STATpathway is frequently observed in primary tumors and leads to increasedangiogenesis, enhanced survival of tumors, and immunosuppression. STATproteins are involved in the development and function of the immunesystem and play a role in maintaining immune tolerance and tumorsurveillance.

STAT proteins are present in the cytoplasm of cells under basalconditions. When activated by tyrosine phosphorylation, STAT proteinsform dimers and translocate to the nucleus where they can bind specificnine base pair sequences in the regulatory regions of target genes,thereby activating transcription. A variety of tyrosine kinases,including polypeptide growth factor receptors, Src family members, andother kinases can catalyze this phosphorylation. While tyrosinephosphorylation is essential for their activation, STAT proteins canalso be phosphorylated on unique serine residues. Although this is notsufficient to induce dimerization and DNA binding, STAT serinephosphorylation modulates the transcriptional response mediated by atyrosine-phosphorylated STAT dimer, and may mediate distinct biologicaleffects (Zhang X, et al. Science 1995; 267:1990-1994; Wen Z, et al. Cell1995; 82:241-250; Kumar A, et al. Science 1997; 278:1630-1632.). STATproteins have been found to function inappropriately in many humanmalignancies (Alvarez J V, et al., Cancer Res 2005; 65(12):5054-62;Frank D A, et al. Cancer Treat. Res. 2003; 115:267-291; Bowman T, et al.Oncogene 2000; 19(21):2474-88).

STAT3 and STAT Modulators

STAT3 is activated in several human tumors, including common epithelialcancers such as cancer of the breast, prostate, lung, pancreas, andovary; hematologic cancers such as multiple myeloma, and acuteleukemias; and diverse tumors such as melanoma and gliomas (Frank D A,et al. Cancer Treat. Res. 2003; 115:267-291). Many of the target genesof STAT3 code for proteins involved in cell survival, cell cycleprogression, differentiation inhibition, invasion, and angiogenesis, allof the essential processes necessary for tumor formation and maintenance(Alvarez J V, et al., Cancer Res 2005; 65(12):5054-62). Inhibition ofSTAT3 function in cancer cells associated with enhanced STAT3 activityleads to a loss of proliferation and survival of the cancer cells (FrankD A. Curr. Cancer Therapy Reviews 2006; 2:57-65). Despite the centralrole that STAT3 plays in these diverse processes in tumor cell biology,loss of STAT3 function in normal adult cells has few if any seriousconsequences, and may in fact decrease the ability of a cell to becometransformed.

An exemplary human STAT3 amino acid sequence is set forth below (SEQ IDNO: 1; GenBank Accession No: AAH14482, Version 1, incorporated herein byreference):

1 maqwnqlqql dtryleqlhq lysdsfpmel rqflapwies qdwayaaske shativfhnl 61lgeidqqysr flqesnvlyq hnlrrikqfl qsrylekpme iarivarclw eesrllqtaa 121taaqqgggan hptaavvtek qqmleghlqd vrkrvqdleq kmkvvenlqd dfdfnyktlk 181sqgdmqdlng nnqsvtrqkm qqlegmltal dqmrrsivse lagllsamey vqktltdeel 241adwkrrqqia ciggppnicl drlenwitsl aesqlqtrqq ikkleelqqk vsykgdpivq 301hrpmleeriv elfrnlmksa fvverucmp mhpdrplvik tgvqfttkvr llvkfpelny 361qlkikvcidk dsgdvaalrg srkfnilgtn tkvmnmeesn ngslsaefkh ltlreqrcgn 421ggrancdasl ivteelhlit fetevyhqgl kidlethslp vvvisnicqm pnawasilwy 481nmltnnpknv nfftkppigt wdqvaevlsw qfssttkrgl sieqlttlae kllgpgvnys 541gcgitwakfc kenmagkgfs fwvwldniid lvkkyilalw negyimgfis kererailst 601kppgtfllrf sesskeggvt ftwvekdisg ktgigsvepy tkqqlnnmsf aeiimgykim 661datnilvspl vylypdipke eafgkycrpe sqehpeadpg saapylktkf icvtpttcsn 721tidlpmsprt ldslmqfgnn gegaepsagg qfesltfdme ltsecatspm

An exemplary human STAT3 nucleic acid sequence is set forth below (SEQID NO: 2; GenBank Accession No: NM_139276, Version 2, incorporatedherein by reference):

1 ggtttccgga gctgcggcgg cgcagactgg gagggggagc cgggggttcc gacgtcgcag 61ccgagggaac aagccccaac cggatcctgg acaggcaccc cggcttggcg ctgtctctcc 121ccctcggctc ggagaggccc ttcggcctga gggagcctcg ccgcccgtcc ccggcacacg 181cgcagccccg gcctctcggc ctctgccgga gaaacagttg ggacccctga ttttagcagg 241atggcccaat ggaatcagct acagcagctt gacacacggt acctggagca gctccatcag 301ctctacagtg acagcttccc aatggagctg cggcagtttc tggccccttg gattgagagt 361caagattggg catatgcggc cagcaaagaa tcacatgcca ctttggtgtt tcataatctc 421ctgggagaga ttgaccagca gtatagccgc ttcctgcaag agtcgaatgt tctctatcag 481cacaatctac gaagaatcaa gcagtttctt cagagcaggt atcttgagaa gccaatggag 541attgcccgga ttgtggcccg gtgcctgtgg gaagaatcac gccttctaca gactgcagcc 601actgcggccc agcaaggggg ccaggccaac caccccacag cagccgtggt gacggagaag 661cagcagatgc tggagcagca ccttcaggat gtccggaaga gagtgcagga tctagaacag 721aaaatgaaag tggtagagaa tctccaggat gactttgatt tcaactataa aaccctcaag 781agtcaaggag acatgcaaga tctgaatgga aacaaccagt cagtgaccag gcagaagatg 841cagcagctgg aacagatgct cactgcgctg gaccagatgc ggagaagcat cgtgagtgag 901ctggcggggc ttttgtcagc gatggagtac gtgcagaaaa ctctcacgga cgaggagctg 961gctgactgga agaggcggca acagattgcc tgcattggag gcccgcccaa catctgccta 1021gatcggctag aaaactggat aacgtcatta gcagaatctc aacttcagac ccgtcaacaa 1081attaagaaac tggaggagtt gcagcaaaaa gtttcctaca aaggggaccc cattgtacag 1141caccggccga tgctggagga gagaatcgtg gagctgttta gaaacttaat gaaaagtgcc 1201tttgtggtgg agcggcagcc ctgcatgccc atgcatcctg accggcccct cgtcatcaag 1261accggcgtcc agttcactac taaagtcagg ttgctggtca aattccctga gttgaattat 1321cagcttaaaa ttaaagtgtg cattgacaaa gactctgggg acgttgcagc tctcagagga 1381tcccggaaat ttaacattct gggcacaaac acaaaagtga tgaacatgga agaatccaac 1441aacggcagcc tctctgcaga attcaaacac ttgaccctga gggagcagag atgtgggaat 1501gggggccgag ccaattgtga tgcttccctg attgtgactg aggagctgca cctgatcacc 1561tttgagaccg aggtgtatca ccaaggcctc aagattgacc tagagaccca ctccttgcca 1621gttgtggtga tctccaacat ctgtcagatg ccaaatgcct gggcgtccat cctgtggtac 1681aacatgctga ccaacaatcc caagaatgta aactttttta ccaagccccc aattggaacc 1741tgggatcaag tggccgaggt cctgagctgg cagttctcct ccaccaccaa gcgaggactg 1801agcatcgagc agctgactac actggcagag aaactcttgg gacctggtgt gaattattca 1861gggtgtcaga tcacatgggc taaattttgc aaagaaaaca tggctggcaa gggcttctcc 1921ttctgggtct ggctggacaa tatcattgac cttgtgaaaa agtacatcct ggccctttgg 1981aacgaagggt acatcatggg ctttatcagt aaggagcggg agcgggccat cttgagcact 2041aagcctccag gcaccttcct gctaagattc agtgaaagca gcaaagaagg aggcgtcact 2101ttcacttggg tggagaagga catcagcggt aagacccaga tccagtccgt ggaaccatac 2161acaaagcagc agctgaacaa catgtcattt gctgaaatca tcatgggcta taagatcatg 2221gatgctacca atatcctggt gtctccactg gtctatctct atcctgacat tcccaaggag 2281gaggcattcg gaaagtattg tcggccagag agccaggagc atcctgaagc tgacccaggt 2341agcgctgccc catacctgaa gaccaagttt atctgtgtga caccaacgac ctgcagcaat 2401accattgacc tgccgatgtc cccccgcact ttagattcat tgatgcagtt tggaaataat 2461ggtgaaggtg ctgaaccctc agcaggaggg cagtttgagt ccctcacctt tgacatggag 2521ttgacctcgg agtgcgctac ctcccccatg tgaggagctg agaacggaag ctgcagaaag 2581atacgactga ggcgcctacc tgcattctgc cacccctcac acagccaaac cccagatcat 2641ctgaaactac taactttgtg gttccagatt ttttttaatc tcctacttct gctatctttg 2701agcaatctgg gcacttttaa aaatagagaa atgagtgaat gtgggtgatc tgcttttatc 2761taaatgcaaa taaggatgtg ttctctgaga cccatgatca ggggatgtgg cggggggtgg 2821ctagagggag aaaaaggaaa tgtcttgtgt tgttttgttc ccctgccctc ctttctcagc 2881agctttttgt tattgttgtt gttgttctta gacaagtgcc tcctggtgcc tgcggcatcc 2941ttctgcctgt ttctgtaagc aaatgccaca ggccacctat agctacatac tcctggcatt 3001gcacttttta accttgctga catccaaata gaagatagga ctatctaagc cctaggtttc 3061tttttaaatt aagaaataat aacaattaaa gggcaaaaaa cactgtatca gcatagcctt 3121tctgtattta agaaacttaa gcagccgggc atggtggctc acgcctgtaa tcccagcact 3181ttgggaggcc gaggcggatc ataaggtcag gagatcaaga ccatcctggc taacacggtg 3241aaaccccgtc tctactaaaa gtacaaaaaa ttagctgggt gtggtggtgg gcgcctgtag 3301tcccagctac tcgggaggct gaggcaggag aatcgcttga acctgagagg cggaggttgc 3361agtgagccaa aattgcacca ctgcacactg cactccatcc tgggcgacag tctgagactc 3421tgtctcaaaa aaaaaaaaaa aaaaaagaaa cttcagttaa cagcctcctt ggtgctttaa 3481gcattcagct tccttcaggc tggtaattta tataatccct gaaacgggct tcaggtcaaa 3541cccttaagac atctgaagct gcaacctggc ctttggtgtt gaaataggaa ggtttaagga 3601gaatctaagc attttagact tttttttata aatagactta ttttcctttg taatgtattg 3661gccttttagt gagtaaggct gggcagaggg tgcttacaac cttgactccc tttctccctg 3721gacttgatct gctgtttcag aggctaggtt gtttctgtgg gtgccttatc agggctggga 3781tacttctgat tctggcttcc ttcctgcccc accctcccga ccccagtccc cctgatcctg 3841ctagaggcat gtctccttgc gtgtctaaag gtccctcatc ctgtttgttt taggaatcct 3901ggtctcagga cctcatggaa gaagaggggg agagagttac aggttggaca tgatgcacac 3961tatggggccc cagcgacgtg tctggttgag ctcagggaat atggttctta gccagtttct 4021tggtgatatc cagtggcact tgtaatggcg tcttcattca gttcatgcag ggcaaaggct 4081tactgataaa cttgagtctg ccctcgtatg agggtgtata cctggcctcc ctctgaggct 4141ggtgactcct ccctgctggg gccccacagg tgaggcagaa cagctagagg gcctccccgc 4201ctgcccgcct tggctggcta gctcgcctct cctgtgcgta tgggaacacc tagcacgtgc 4261tggatgggct gcctctgact cagaggcatg gccggatttg gcaactcaaa accaccttgc 4321ctcagctgat cagagtttct gtggaattct gtttgttaaa tcaaattagc tggtctctga 4381attaaggggg agacgacctt ctctaagatg aacagggttc gccccagtcc tcctgcctgg 4441agacagttga tgtgtcatgc agagctctta cttctccagc aacactcttc agtacataat 4501aagcttaact gataaacaga atatttagaa aggtgagact tgggcttacc attgggttta 4561aatcataggg acctagggcg agggttcagg gcttctctgg agcagatatt gtcaagttca 4621tggccttagg tagcatgtat ctggtcttaa ctctgattgt agcaaaagtt ctgagaggag 4681ctgagccctg ttgtggccca ttaaagaaca gggtcctcag gccctgcccg cttcctgtcc 4741actgccccct ccccatcccc agcccagccg agggaatccc gtgggttgct tacctaccta 4801taaggtggtt tataagctgc tgtcctggcc actgcattca aattccaatg tgtacttcat 4861agtgtaaaaa tttatattat tgtgaggttt tttgtctttt tttttttttt ttttttttgg 4921tatattgctg tatctacttt aacttccaga aataaacgtt atataggaac cgtaaaaa

The following compounds are STAT3 inhibitors: pyrimethamine, atovaquone,pimozide, guanabenz acetate, alprenolol hydrochloride, nifuroxazide,solanine alpha, fluoxetine hydrochloride, ifosfamide, pyrvinium pamoate,moricizine hydrochloride, 3,3′-oxybis[tetrahydrothiophene,1,1,1′,1′-tetraoxide],3-(1,3-benzodioxol-5-yl)-1,6-dimethyl-pyrimido[5,4-e]-1,2,4-triazine-5,7(-1H,6H)-dione,2-(1,8-Naphthyridin-2-yl)phenol,3-(2-hydroxyphenyl)-3-phenyl-N,N-dipropylpropanamide as well as anyderivatives of these compounds or analogues thereof. These compounds arecommercially available through various sources.

Another exemplary STAT3 inhibitor includes JSI-124. Cucurbitacin I(JSI-124) is a selective inhibitor of the janus kinase 2/signaltransducer and activator of transcription 3 (JAK2/STAT3) signalingpathway with anti-proliferative and anti-tumor properties. The structureof JSI-124 (cucurbitacin I) is set forth below (Blaskovich et al., 2003Cancer Res., 63(6): 1270-1279; incorporated herein by reference).

Single-cell RNA-sequencing of ovarian cancer in patients and PDX modelsguides strategies to overcome platinum-resistance.

Ovarian cancer is one of the leading causes of cancer-related deaths inwomen. Many women with OvCa experience relapse characterized by minimalresidual disease following platinum-based chemotherapy, development ofmalignant abdominal fluid (ascites) with tumor spheroids, and extensivetumor heterogeneity: features not easily captured by current genomicprofiling approaches. Resistance to platinum-based therapies anddevelopment of ascites constitutes the major life-limiting factor inwomen with ovarian cancer, and the underlying mechanisms remain unknown.To capture inherent heterogeneity and complexity of this ecosystem,single-cell RNA-sequencing was applied to ˜17,500 cells, isolated from11 OvCa patients, to map the cellular ascites ecosystem. Significantinter-individual variability in the cellular composition of ascites andthe immunomodulatory functions of non-malignant cells, such asinterleukin-6 producing cancer-associated fibroblasts (CAFs), wasobserved. Previously described “immunoreactive” and “mesenchymal”expression subtypes of OvCa are rather reflective of infiltration withimmune cells and CAFs, respectively. A common feature of resistance, andalso a driver of heterogeneity among malignant cells, is the cancercell-autonomous expression of inflammatory pathways, including theJAK/STAT-pathway. To systematically interrogate this observationregarding platinum-resistance, PDX models were treated with carboplatin,and scRNA-seq was performed on pre-treatment, at the time of minimalresidual disease (MRD) and on relapse. Expression of JAK/STAT pathwaycomponents were found to be expressed in MRD/relapse. Specifically, acommon feature of MRD/relapsed cells was expression of interferonsignaling and robust expression of JAK/STAT pathway components,indicating an important role of this pathway in platinum-resistance.JAK/STAT-inhibition (using JSI-124 at nano-molar concentrations) ofplatinum-resistant patient-derived ex vivo cultures led to inhibition offormation and invasion of spheroids through a mesothelial monolayer, andresulted in disintegration of spheroids. JAK/STAT-inhibition efficientlyovercame platinum-resistance in patient-derived ex vivo cultures,inhibits metastatic potential and invasion, prevents formation ofmalignant ascites and leads to tumor regression in PDX models. Theresults presented herein indicate that IP-injected JSI-124 preventedformation and disrupted malignant ascites and sub-cutaneous tumors PDXmodels, indicating a potentially unique therapeutic niche for patientswith advanced OvCa, in which IP chemo is frequently administered. Insummary, the ecosystem of platinum-resistant ovarian cancer usingscRNA-seq in patients and PDX models was mapped herein, and thisecosystem provides a therapeutic strategy for platinum-resistantdisease.

Tumors are multicellular assemblies that encompass cells with distinctgenotypic and phenotypic states. Thus, single-cell RNA-seq was appliedto ovarian cancer samples. Overall, the analysis described in detailbelow unravels the cellular ecosystem of ovarian tumors and shows thatsingle cell genomics offers new insights with implications for bothtargeted and immune therapies.

Described herein is the first comprehensive single-cell transcriptomeanalysis of malignant ascites in platinum-resistant ovarian cancerpatients. Analysis reveals significant variability of the cellularcomposition and diversity among malignant and non-malignant cells inascites. In line with prior single-cell studies in melanoma and gliomasolid tumors, the patient of origin primarily determined heterogeneityamong cancer cells. However, in contrast to these other cancers, asignificant inter-individual heterogeneity of non-malignant cells wasobserved, both in their abundance and transcriptional cell states. Thisvariability was particularly distinct within some non-malignant cellpopulations, such as macrophages and cancer-associated fibroblasts(CAFs). While CAFs expressed common genes, such as complement factors,populations were identified with discrete expression patterns ofimmunomodulatory genes, including inflammatory and immunosuppressivecytokines, such as IL-6 and IL-10, respectively. Elevated levels of IL-6in the ascites have previously been associated with poor clinicaloutcomes; however, the source of IL-6 has been unknown. While severalcell types express IL-6, including cancer cells, CAFs showed thestrongest expression. Thus, non-malignant cells significantly contributeto shaping the ascites milieu.

Resistance to platinum-based chemotherapy invariably develops inpatients with advanced ovarian cancer and is frequently associated withthe development of malignant abdominal fluid (ascites). The mechanismsof drug resistance are poorly understood. A recent whole-genomesequencing study of 92 chemoresistant patients revealed frequent TP53mutations, but no recurrent or potentially actionable oncogenic drivers(Patch et al., 2015 Nature, 521:489-494). This study emphasized thepreviously described vast genomic heterogeneity (T. C. G. A. R. Network,2011 Nature, 474:609-615) and adaptability of ovarian cancer,underscoring the need for additional, high-resolution phenotypicanalysis of platinum-resistance. In this study, the first comprehensivesingle-cell transcriptome analysis of malignant ascites in ovariancancer patients was performed. The analysis reveals significantvariability of the cellular composition and diversity among malignantand non-malignant cells in ascites. In line with prior single-cellstudies in melanoma and glioma solid tumors, the patient of originprimarily determined heterogeneity among cancer cells (Tirosh et al.,2016 Science, 352:189-196). However, a significant inter-individualheterogeneity of non-malignant cells, both in their abundance andtranscriptional cell states, was observed. This variability wasparticularly distinct within some non-malignant cell populations, suchas macrophages and cancer-associated fibroblasts (CAFs). While CAFsexpressed common genes, such as complement factors, populations withdiscrete expression patterns of immunomodulatory genes were identified,including inflammatory and immunosuppressive cytokines, such as IL-6 andIL-10, respectively. Elevated levels of IL-6 in the ascites havepreviously been associated with poor clinical outcomes (Lane et al.,2011 BMC Cancer, 11:210; Kolomeyevskaya et al., 2015 Gynecol. Oncol.,138:352-357); however, the proposed source of IL-6 has been variableacross studies. While several cell types express IL-6, including cancercells, CAFs showed the strongest expression. Together, these initialinsights suggest that non-malignant cells significantly contribute toshaping the ascites milieu. Furthermore, the strength of the single cellprofiling techniques detailed above was crucial in later identifying asub-population of CAFs as a major source of IL6 within the ascitesecosystem.

Using previously described methods (Tirosh et al., 2016 Science,352:189-196), large-scale CNVs were inferred. In line with prior studies(Patch et al., 2015 Nature, 521:489-494), CNVs were not identified incancer cell sub-populations that account for resistant cells, but ratherminimal changes in the CNV patterns in those patients where sequentialsamples were available. The study also investigated whether ovariancancer subtypes described by TCGA (T.C.G.A.R. Network, 2011 Nature,474:609-615) contribute to variability among patients and samples.Surprisingly, cancer cells from all patients strongly expressed the“differentiated” subtype program and cells from one patient alsoexpressed the “proliferative” subtype. In contrast, the previouslydescribed “mesenchymal” and “immunoreactive” subtypes were not expressedwithin cancer cells, but were rather reflecting programs expressed byCAFs and macrophages, respectively. This result indicates that themesenchymal and immunoreactive subtypes are infiltrated with CAFs orimmune cells, respectively, and highlight how single-cell profilingresolves the multicellular composition of cancers and identifies thecell-type specific origin of transcriptional programs. A similarobservation of “transcriptional mimicry” was made in colorectal cancer,where two studies identified transcriptomes of CAFs as source for atumor sub-type (Isella et al., 2015 Nat. Genet., 47:312-319; Calon etal., 2015 Nat. Genet. 47: 320-329).

Previously identified were recurrent transcriptional cell statescharacterized by high expression of AXL and low expression oflineage-markers, such as MITF, conferring resistance to drug therapiesin melanoma (Tirosh et al., 2016 Science, 352:189-196). In contrast,results did not find a consistent platinum-resistance program acrosspatients with ovarian cancer. While several factors contribute to thisobservation, two key differences between ovarian cancer and othercancers are critical: (i) there are no highly recurrent targetableoncogenic drivers and (ii) effects of platinum-based therapies arepleiotropic and provoke variable cellular responses, and thereforevariable mechanisms of resistance. However, a consistent observationacross platinum-resistant patients was the cancer cell-autonomousexpression of major inflammatory pathways, including the JAK/STATpathway, interferon signaling and expression of several inflammatorycytokines, such as tumor necrosis factor (TNF) alpha. In particular,positive regulatory circuits involving the JAK/STAT pathway have beenshown to promote malignant transformation and maintenance in breastcancer. Moreover, prior studies suggested that regulatory circuitsinvolving the JAK/STAT pathway promotes malignant growth in ovariancancer (Kulbe et al., 2012 Cancer Res., 72:66-75; Wen et al., 2014 Mol.Cancer Ther. 13:3037-3048; Saini et al., 2017 Oncogene, 36:168-181). Toobtain a more controlled perspective on changes that occur over timefollowing platinum chemotherapy, scRNA-seq was performed on cells fromPDX models obtained a) before treatment, b) at the time of maximalresponse (or minimal residual disease, MRD) to carboplatin and c) at thetime of relapse. Similar to the findings in ascites, residual orrelapsed disease following platinum treatment was associated withdistinct profiles across models derived from different patients, and arecurrent program within MRD/relapse across models was not found.However, consistent with findings in clinically platinum-resistantpatients, MRD/relapse samples exhibited specific expression of severalimmune related genes, including key components of the JAK/STAT pathway,interferon signaling, the CXCL-12 receptor C-X-C chemokine receptor type4 (CXCR4) and other inflammatory genes. Together with the extensivevariability in immune-related programs and the widespread expression ofinterleukin (IL)-6, these results suggested that ovarian cancer cellsdepend on upstream regulators of inflammatory pathways and suggested arole of these pathways in platinum-resistance. The consistent expressionin PDX models independent of prior platinum therapy indicates that theseprograms are relevant for both platinum-naïve and platinum-resistantdisease.

Among other ligands, IL6 and C-X-C motif chemokine ligand 12 (CXCL-12)are strong upstream activators of JAK/STAT signaling, and were stronglyexpressed by non-malignant cells in platinum-resistant cells. TheCXLC-12 receptor CXCR-4 was furthermore strongly upregulated in aportion of cancer cells from ascites and PDX-derived cells at MRD andrelapse, indicating its role in platinum-resistance, and has beencorrelated with poor prognosis (Popple et al., 2012 Br. J. Cancer.,106:1306-1313). JAK/STAT pathway activation has been described invarious solid tumors. Clinical assessment by immunohistochemistry (IHC)of STAT3 or phosphorylated STAT3 (pSTAT3) yielded variable prognostic orpredictive roles across cancers, including breast (Sonnenblick et al.,2013 Breast Cancer Res. Treat., 138:407-413), prostate (Mirtti et al.,2013 Hum. Pathol., 44:310-319), lung (Y. H. Xu and S. Lu, 2014 Eur. J.Surg. Oncol. J. Eur. Soc. Surg. Oncol. Br. Assoc. Surg. Oncol.,40:311-317), head and neck (Pectasides et al., 2010 Clin. Cancer Res.Off. J. Am. Assoc. Cancer Res., 16:2427-2434), renal cell cancer(Horiguchi et al., 2002 J. Urol., 168:762-765), melanoma (Messina etal., 2008 Cancer control J. Moffitt Cancer Cent., 15:196-201) andglioblastoma (Birner et al., 2010 J. Neurooncol, 100:339-343). Inmyeloproliferative disorders, such as essential thrombocytemia (ET) andpolycythemia vera (PV), treatment with JAK/STAT inhibitors (such asruxolitinib) results in improved disease control (W. Vainchenker and S.N. Constantinescu, 2013 Oncogene, 32:26012613); interestingly, theseagents are effective irrespective of the JAK2 mutation status,indicating that JAK/STAT inhibition is a feasible therapeutic avenue inother contexts with known JAK/STAT pathway activation. Previous workindicates increased IL-6-dependent activation of JAK/STAT signaling incells isolated from patient-derived ascites (Saini et al., 2017Oncogene, 36:168-18). Both autocrine and paracrine IL-6 signaling hasbeen described in ovarian cancer, and the IL-6 receptor and soluble IL-6receptor appear to modulate ovarian cancer growth. IL-6 is alsoimplicated in a feedback circuit with paraneoplastic thrombocytosis thatenhances ovarian cancer growth (Stone et al., 2012 N. Engl. J. Med.,366: 610-618). Targeting IL-6 with an anti-IL-6 antibody siltuximab(Coward et al., 2011 Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res.,17:6083-6096) or an anti-IL6-R antibody tocilizumab (Dijkgraaf et al.,2015 Ann. Oncol. Off. J. Eur. Soc. Med. Oncol., 26: 2141-2149) has beenexplored in phase I trials in ovarian cancer.

Despite these intriguing results, the role of JAK/STAT signaling inplatinum-resistance and its potential as a therapeutic target in ovariancancer are incompletely understood. JSI-124 (also known as CucurbitacinI) has previously been identified as a selective JAK/STAT inhibitoramong a library of STAT3 inhibitors, and was safely administered to lungcancer and melanoma mouse models (Blaskovich et al., 2003 Cancer Res.,63: 1270-1279). Therefore, the effects of JSI-124 on several criticalsteps of ovarian cancer pathogenesis and drug resistance were examined.The results presented herein show that treatment of ovarian cancer celllines and primary ovarian cancer ascites cells with JSI-124 can, i)prevent or reduce the formation of spheroids in a dose-dependent mannerin vitro, ii) inhibit invasion of spheroids through a mesothelialmonolayer ex vivo and in vitro, and iii) effectively and selectivelykill cancer cells in formed spheroids derived from patients withplatinum-resistance, that are resistant to other federal drugadministration (FDA)-approved chemotherapies for ovarian cancer. In aBRCA-wild type (WT) PDX model, IP administered JSI-124 effectively i)prevents formation of malignant ascites, ii) kills established malignantascites iii) prevents growth of SC injected tumors, and iv) leads toregression of established SC tumors. Together, these results indicatethat IP-injected JSI-124 has local and systemic activity against ovariancancer. IP therapy with cisplatin (in combination with IV paclitaxel) isa frequently used therapeutic modality in patients with stage III/IVovarian cancer and improves progression-free and overall survival insome settings (Armstrong et al., 2006 N. Engl. J. Med., 354: 34-43).Therefore, IP drug administration is a unique therapeutic niche in thetreatment of ovarian cancer. While toxicities require furtherinvestigation, the use of JSI-124 as adjuvant or neoadjuvant therapyand/or in the context of platinum resistance represents a feasibletherapeutic strategy. Indeed, a recently launched phase I/II trial(NCT02713386) examines the safety and efficacy of JAK/STAT inhibition(using ruxolitinib) in combination with carboplatin and paclitaxel inthe neo-adjuvant and adjuvant setting in patients with advancedgynecologic cancers, including ovarian cancer.

In summary, provided herein are a comprehensive map of single-celltranscriptomes of platinum-resistant ascites from patients with ovariancancer and a sequential profiling of PDX models following platinumtreatment. These data comprise a significant data resource toinvestigate cellular heterogeneity and platinum resistance in ovariancancer. The results highlight the importance of cancer cell-autonomousexpression of various inflammatory pathways. Results from these analysesguided subsequent identification of activation of the JAK/STAT pathwayas a recurrent feature of ovarian cancer cells and associatednon-malignant cells, particularly in the context of platinum resistance.In other words, results for these analyses guided identification of aputative mechanism of resistance, i.e., the activation of the JAK/STATpathway. Further, results herein show that JAK/STAT inhibition usingJSI-124, when compared to other routinely used agents in ovarian cancer,shows superior activity abrogating several key steps in ovarian cancermetastatic pathogenesis and inhibiting established malignant ascites andtumors ex vivo and in vivo. This analysis highlights the potential ofsingle-cell genomic studies to provide rationale for the development oftherapeutic strategies that are feasible for translation into clinicalpatient care.

Pharmaceutical Therapeutics

For therapeutic uses, the compositions or agents described herein may beadministered systemically, for example, formulated in apharmaceutically-acceptable buffer such as physiological saline.Preferable routes of administration include, for example, subcutaneous,intravenous, interperitoneal, intramuscular, or intradermal injectionsthat provide continuous, sustained levels of the drug in the patient.Treatment of human patients or other animals will be carried out using atherapeutically effective amount of a therapeutic identified herein in aphysiologically-acceptable carrier. Suitable carriers and theirformulation are described, for example, in Remington's PharmaceuticalSciences by E. W. Martin. The amount of the therapeutic agent to beadministered varies depending upon the manner of administration, the ageand body weight of the patient, and with the clinical symptoms of theneoplasia. Generally, amounts will be in the range of those used forother agents used in the treatment of other diseases associated withneoplasia, although in certain instances lower amounts will be neededbecause of the increased specificity of the compound. For example, atherapeutic compound is administered at a dosage that is cytotoxic to aneoplastic cell.

Formulation of Pharmaceutical Compositions

Human dosage amounts can initially be determined by extrapolating fromthe amount of compound used in mice, as a skilled artisan recognizes itis routine in the art to modify the dosage for humans compared to animalmodels. In certain embodiments, it is envisioned that the dosage mayvary from between about 1 μg compound/Kg body weight to about 5000 mgcompound/Kg body weight; or from about 5 mg/Kg body weight to about 4000mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kgbody weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg bodyweight; or from about 100 mg/Kg body weight to about 1000 mg/Kg bodyweight; or from about 150 mg/Kg body weight to about 500 mg/Kg bodyweight. In other cases, this dose may be about 1, 5, 10, 25, 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000,4500, or 5000 mg/Kg body weight. In other aspects, it is envisaged thatdoses may be in the range of about 5 mg compound/Kg body to about 20 mgcompound/Kg body. In other embodiments, the doses may be about 8, 10,12, 14, 16 or 18 mg/Kg body weight. Of course, this dosage amount may beadjusted upward or downward, as is routinely done in such treatmentprotocols, depending on the results of the initial clinical trials andthe needs of a particular patient.

In some cases, the compound or composition of the invention isadministered at a dose that is lower than the human equivalent dosage(HED) of the no observed adverse effect level (NOAEL) over a period ofthree months, four months, six months, nine months, 1 year, 2 years, 3years, 4 years or more. The NOAEL, as determined in animal studies, isuseful in determining the maximum recommended starting dose for humanclinical trials. For instance, the NOAELs can be extrapolated todetermine human equivalent dosages. Typically, such extrapolationsbetween species are conducted based on the doses that are normalized tobody surface area (i.e., mg/m²). In specific embodiments, the NOAELs aredetermined in mice, hamsters, rats, ferrets, guinea pigs, rabbits, dogs,primates, primates (monkeys, marmosets, squirrel monkeys, baboons),micropigs or minipigs. For a discussion on the use of NOAELs and theirextrapolation to determine human equivalent doses, see Guidance forIndustry Estimating the Maximum Safe Starting Dose in Initial ClinicalTrials for Therapeutics in Adult Healthy Volunteers, U.S. Department ofHealth and Human Services Food and Drug Administration Center for DrugEvaluation and Research (CDER), Pharmacology and Toxicology, July 2005,incorporated herein by reference.

The amount of a compound of the invention used in the prophylacticand/or therapeutic regimens which will be effective in the prevention,treatment, and/or management of cancer can be based on the currentlyprescribed dosage of the compound as well as assessed by methodsdisclosed herein and known in the art. The frequency and dosage willvary also according to factors specific for each patient depending onthe specific compounds administered, the severity of the cancerouscondition, the route of administration, as well as age, body, weight,response, and the past medical history of the patient. For example, thedosage of a compound of the invention which will be effective in thetreatment, prevention, and/or management of cancer can be determined byadministering the compound to an animal model such as, e.g., the animalmodels disclosed herein or known to those skilled in the art. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges.

In some aspects, the prophylactic and/or therapeutic regimens comprisetitrating the dosages administered to the patient so as to achieve aspecified measure of therapeutic efficacy. Such measures include areduction in the cancer cell population in the patient.

In certain cases, the dosage of the compound of the invention in theprophylactic and/or therapeutic regimen is adjusted so as to achieve areduction in the number or amount of cancer cells found in a testspecimen extracted from a patient after undergoing the prophylacticand/or therapeutic regimen, as compared with a reference sample. Here,the reference sample is a specimen extracted from the patient undergoingtherapy, wherein the specimen is extracted from the patient at anearlier time point. In one aspect, the reference sample is a specimenextracted from the same patient, prior to receiving the prophylacticand/or therapeutic regimen. For example, the number or amount of cancercells in the test specimen is at least 2%, 5%, 10%, 15%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or 99% lower than in the reference sample.

In some cases, the dosage of the compound of the invention in theprophylactic and/or therapeutic regimen is adjusted so as to achieve anumber or amount of cancer cells that falls within a predeterminedreference range. In these embodiments, the number or amount of cancercells in a test specimen is compared with a predetermined referencerange.

In other embodiments, the dosage of the compound of the invention inprophylactic and/or therapeutic regimen is adjusted so as to achieve areduction in the number or amount of cancer cells found in a testspecimen extracted from a patient after undergoing the prophylacticand/or therapeutic regimen, as compared with a reference sample, whereinthe reference sample is a specimen is extracted from a healthy,noncancer-afflicted patient. For example, the number or amount of cancercells in the test specimen is at least within 60%, 50%, 40%, 30%, 20%,15%, 10%, 5%, or 2% of the number or amount of cancer cells in thereference sample.

In treating certain human patients having solid tumors, extractingmultiple tissue specimens from a suspected tumor site may proveimpracticable. In these cases, the dosage of the compounds of theinvention in the prophylactic and/or therapeutic regimen for a humanpatient is extrapolated from doses in animal models that are effectiveto reduce the cancer population in those animal models. In the animalmodels, the prophylactic and/or therapeutic regimens are adjusted so asto achieve a reduction in the number or amount of cancer cells found ina test specimen extracted from an animal after undergoing theprophylactic and/or therapeutic regimen, as compared with a referencesample. The reference sample can be a specimen extracted from the sameanimal, prior to receiving the prophylactic and/or therapeutic regimen.In specific embodiments, the number or amount of cancer cells in thetest specimen is at least 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50% or 60%lower than in the reference sample. The doses effective in reducing thenumber or amount of cancer cells in the animals can be normalized tobody surface area (e.g., mg/m²) to provide an equivalent human dose.

The prophylactic and/or therapeutic regimens disclosed herein compriseadministration of compounds of the invention or pharmaceuticalcompositions thereof to the patient in a single dose or in multipledoses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more doses).

In one aspect, the prophylactic and/or therapeutic regimens compriseadministration of the compounds of the invention or pharmaceuticalcompositions thereof in multiple doses. When administered in multipledoses, the compounds or pharmaceutical compositions are administeredwith a frequency and in an amount sufficient to prevent, treat, and/ormanage the condition. For example, the frequency of administrationranges from once a day up to about once every eight weeks. In anotherexample, the frequency of administration ranges from about once a weekup to about once every six weeks. In another example, the frequency ofadministration ranges from about once every three weeks up to about onceevery four weeks.

Generally, the dosage of a compound of the invention administered to asubject to prevent, treat, and/or manage cancer is in the range of 0.01to 500 mg/kg, e.g., in the range of 0.1 mg/kg to 100 mg/kg, of thesubject's body weight. For example, the dosage administered to a subjectis in the range of 0.1 mg/kg to 50 mg/kg, or 1 mg/kg to 50 mg/kg, of thesubject's body weight, more preferably in the range of 0.1 mg/kg to 25mg/kg, or 1 mg/kg to 25 mg/kg, of the patient's body weight. In anotherexample, the dosage of a compound of the invention administered to asubject to prevent, treat, and/or manage cancer in a patient is 500mg/kg or less, preferably 250 mg/kg or less, 100 mg/kg or less, 95 mg/kgor less, 90 mg/kg or less, 85 mg/kg or less, 80 mg/kg or less, 75 mg/kgor less, 70 mg/kg or less, 65 mg/kg or less, 60 mg/kg or less, 55 mg/kgor less, 50 mg/kg or less, 45 mg/kg or less, 40 mg/kg or less, 35 mg/kgor less, 30 mg/kg or less, 25 mg/kg or less, 20 mg/kg or less, 15 mg/kgor less, 10 mg/kg or less, 5 mg/kg or less, 2.5 mg/kg or less, 2 mg/kgor less, 1.5 mg/kg or less, or 1 mg/kg or less of a patient's bodyweight.

In another example, the dosage of a compound of the inventionadministered to a subject to prevent, treat, and/or manage cancer in apatient is a unit dose of 0.1 to 50 mg, 0.1 mg to 20 mg, 0.1 mg to 15mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg,0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg,0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

In another example, the dosage of a compound of the inventionadministered to a subject to prevent, treat, and/or manage cancer in apatient is in the range of 0.01 to 10 g/m², and more typically, in therange of 0.1 g/m² to 7.5 g/m², of the subject's body weight. Forexample, the dosage administered to a subject is in the range of 0.5g/m² to 5 g/m², or 1 g/m² to 5 g/m² of the subject's body's surfacearea.

In another example, the prophylactic and/or therapeutic regimencomprises administering to a patient one or more doses of an effectiveamount of a compound of the invention, wherein the dose of an effectiveamount achieves a plasma level of at least 0.1 μg/mL, at least 0.5μg/mL, at least 1 μg/mL, at least 2 μg/mL, at least 5 μg/mL, at least 6μg/mL, at least 10 μg/mL, at least 15 μg/mL, at least 20 μg/mL, at least25 μg/mL, at least 50 μg/mL, at least 100 μg/mL, at least 125 μg/mL, atleast 150 μg/mL, at least 175 μg/mL, at least 200 μg/mL, at least 225μg/mL, at least 250 μg/mL, at least 275 μg/mL, at least 300 μg/mL, atleast 325 μg/mL, at least 350 μg/mL, at least 375 μg/mL, or at least 400μg/mL of the compound of the invention.

In another example, the prophylactic and/or therapeutic regimencomprises administering to a patient a plurality of doses of aneffective amount of a compound of the invention, wherein the pluralityof doses maintains a plasma level of at least 0.1 μg/mL, at least 0.5μg/mL, at least 1 μg/mL, at least 2 μg/mL, at least 5 μg/mL, at least 6μg/mL, at least 10 μg/mL, at least 15 μg/mL, at least 20 μg/mL, at least25 μg/mL, at least 50 μg/mL, at least 100 μg/mL, at least 125 μg/mL, atleast 150 μg/mL, at least 175 μg/mL, at least 200 μg/mL, at least 225μg/mL, at least 250 μg/mL, at least 275 μg/mL, at least 300 μg/mL, atleast 325 μg/mL, at least 350 μg/mL, at least 375 μg/mL, or at least 400μg/mL of the compound of the invention for at least 1 day, 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 monthsor 36 months.

In other embodiments, the prophylactic and/or therapeutic regimencomprises administering to a patient a plurality of doses of aneffective amount of a compound of the invention, wherein the pluralityof doses maintains a plasma level of at least 0.1 μg/mL, at least 0.5μg/mL, at least 1 μg/mL, at least 2 μg/mL, at least 5 μg/mL, at least 6μg/mL, at least 10 μg/mL, at least 15 μg/mL, at least 20 μg/mL, at least25 μg/mL, at least 50 μg/mL, at least 100 μg/mL, at least 125 μg/mL, atleast 150 μg/mL, at least 175 μg/mL, at least 200 μg/mL, at least 225μg/mL, at least 250 μg/mL, at least 275 μg/mL, at least 300 μg/mL, atleast 325 μg/mL, at least 350 μg/mL, at least 375 μg/mL, or at least 400μg/mL of the compound of the invention for at least 1 day, 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 monthsor 36 months.

Combination Therapy

In one example, the active compounds are administered in combinationtherapy, i.e., combined with other agents, e.g., therapeutic agents,that are useful for treating pathological conditions or disorders, suchas various forms of cancer. The term “in combination” in this contextmeans that the agents are given substantially contemporaneously, eithersimultaneously or sequentially. If given sequentially, at the onset ofadministration of the second compound, the first of the two compounds isin some cases still detectable at effective concentrations at the siteof treatment.

The administration of a compound or a combination of compounds for thetreatment of a neoplasia may be by any suitable means that results in aconcentration of the therapeutic that, combined with other components,is effective in ameliorating, reducing, or stabilizing a neoplasia. Thecompound may be contained in any appropriate amount in any suitablecarrier substance, and is generally present in an amount of 1-95% byweight of the total weight of the composition. The composition may beprovided in a dosage form that is suitable for parenteral (e.g.,subcutaneously, intravenously, intramuscularly, or intraperitoneally)administration route. The pharmaceutical compositions may be formulatedaccording to conventional pharmaceutical practice (see, e.g., Remington:The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro,Lippincott Williams & Wilkins, 2000 and Encyclopedia of PharmaceuticalTechnology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, MarcelDekker, New York).

Accordingly, in some examples, the prophylactic and/or therapeuticregimen comprises administration of a compound of the invention incombination with one or more additional anticancer therapeutics. In oneexample, the dosages of the one or more additional anticancertherapeutics used in the combination therapy is lower than those whichhave been or are currently being used to prevent, treat, and/or managecancer. The recommended dosages of the one or more additional anticancertherapeutics currently used for the prevention, treatment, and/ormanagement of cancer can be obtained from any reference in the artincluding, but not limited to, Hardman et al., eds., Goodman & Gilman'sThe Pharmacological Basis Of Basis Of Therapeutics, 10th ed.,McGraw-Hill, New York, 2001; Physician's Desk Reference (60th ed.,2006), which is incorporated herein by reference in its entirety.

The compound of the invention and the one or more additional anticancertherapeutics can be administered separately, simultaneously, orsequentially. In various aspects, the compound of the invention and theadditional anticancer therapeutic are administered less than 5 minutesapart, less than 30 minutes apart, less than 1 hour apart, at about 1hour apart, at about 1 to about 2 hours apart, at about 2 hours to about3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hoursto about 5 hours apart, at about 5 hours to about 6 hours apart, atabout 6 hours to about 7 hours apart, at about 7 hours to about 8 hoursapart, at about 8 hours to about 9 hours apart, at about 9 hours toabout 10 hours apart, at about 10 hours to about 11 hours apart, atabout 11 hours to about 12 hours apart, at about 12 hours to 18 hoursapart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hoursto 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hoursapart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hoursto 96 hours apart, or 96 hours to 120 hours part. In another example,two or more anticancer therapeutics are administered within the samepatient visit.

In certain aspects, the compound of the invention and the additionalanticancer therapeutic are cyclically administered. Cycling therapyinvolves the administration of one anticancer therapeutic for a periodof time, followed by the administration of a second anticancertherapeutic for a period of time and repeating this sequentialadministration, i.e., the cycle, in order to reduce the development ofresistance to one or both of the anticancer therapeutics, to avoid orreduce the side effects of one or both of the anticancer therapeutics,and/or to improve the efficacy of the therapies. In one example, cyclingtherapy involves the administration of a first anticancer therapeuticfor a period of time, followed by the administration of a secondanticancer therapeutic for a period of time, optionally, followed by theadministration of a third anticancer therapeutic for a period of timeand so forth, and repeating this sequential administration, i.e., thecycle in order to reduce the development of resistance to one of theanticancer therapeutics, to avoid or reduce the side effects of one ofthe anticancer therapeutics, and/or to improve the efficacy of theanticancer therapeutics.

In another example, the anticancer therapeutics are administeredconcurrently to a subject in separate compositions. The combinationanticancer therapeutics of the invention may be administered to asubject by the same or different routes of administration.

When a compound of the invention and the additional anticancertherapeutic are administered to a subject concurrently, the term“concurrently” is not limited to the administration of the anticancertherapeutics at exactly the same time, but rather, it is meant that theyare administered to a subject in a sequence and within a time intervalsuch that they can act together (e.g., synergistically to provide anincreased benefit than if they were administered otherwise). Forexample, the anticancer therapeutics may be administered at the sametime or sequentially in any order at different points in time; however,if not administered at the same time, they should be administeredsufficiently close in time so as to provide the desired therapeuticeffect, preferably in a synergistic fashion. The combination anticancertherapeutics of the invention can be administered separately, in anyappropriate form and by any suitable route. When the components of thecombination anticancer therapeutics are not administered in the samepharmaceutical composition, it is understood that they can beadministered in any order to a subject in need thereof. For example, acompound of the invention can be administered prior to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of theadditional anticancer therapeutic, to a subject in need thereof. Invarious aspects, the anticancer therapeutics are administered 1 minuteapart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hoursto 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hoursapart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hoursto 12 hours apart, no more than 24 hours apart or no more than 48 hoursapart. In one example, the anticancer therapeutics are administeredwithin the same office visit. In another example, the combinationanticancer therapeutics of the invention are administered at 1 minute to24 hours apart.

Release of Pharmaceutical Compositions

Pharmaceutical compositions according to the invention may be formulatedto release the active compound substantially immediately uponadministration or at any predetermined time or time period afteradministration. The latter types of compositions are generally known ascontrolled release formulations, which include (i) formulations thatcreate a substantially constant concentration of the drug within thebody over an extended period of time; (ii) formulations that after apredetermined lag time create a substantially constant concentration ofthe drug within the body over an extended period of time; (iii)formulations that sustain action during a predetermined time period bymaintaining a relatively, constant, effective level in the body withconcomitant minimization of undesirable side effects associated withfluctuations in the plasma level of the active substance (sawtoothkinetic pattern); (iv) formulations that localize action by, e.g.,spatial placement of a controlled release composition adjacent to or incontact with the thymus; (v) formulations that allow for convenientdosing, such that doses are administered, for example, once every one ortwo weeks; and (vi) formulations that target a neoplasia by usingcarriers or chemical derivatives to deliver the therapeutic agent to aparticular cell type (e.g., neoplastic cell). For some applications,controlled release formulations obviate the need for frequent dosingduring the day to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the compound in question. In one example, controlledrelease is obtained by appropriate selection of various formulationparameters and ingredients, including, e.g., various types of controlledrelease compositions and coatings. Thus, the therapeutic is formulatedwith appropriate excipients into a pharmaceutical composition that, uponadministration, releases the therapeutic in a controlled manner.Examples include single or multiple unit tablet or capsule compositions,oil solutions, suspensions, emulsions, microcapsules, microspheres,molecular complexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

The pharmaceutical composition may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.Formulations can be found in Remington: The Science and Practice ofPharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in the form of a solution, a suspension, an emulsion,an infusion device, or a delivery device for implantation, or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active agent that reduces orameliorates a neoplasia, the composition may include suitableparenterally acceptable carriers and/or excipients. The activetherapeutic agent(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions according to theinvention may be in the form suitable for sterile injection. To preparesuch a composition, the suitable active antineoplastic therapeutic(s)are dissolved or suspended in a parenterally acceptable liquid vehicle.Among acceptable vehicles and solvents that may be employed are water,water adjusted to a suitable pH by addition of an appropriate amount ofhydrochloric acid, sodium hydroxide or a suitable buffer,1,3-butanediol, Ringer's solution, and isotonic sodium chloride solutionand dextrose solution. The aqueous formulation may also contain one ormore preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).In cases where one of the compounds is only sparingly or slightlysoluble in water, a dissolution enhancing or solubilizing agent can beadded, or the solvent may include 10-60% w/w of propylene glycol.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the active drugmay be incorporated in biocompatible carriers, liposomes, nanoparticles,implants, or infusion devices.

Materials for use in the preparation of microspheres and/ormicrocapsules are, e.g., biodegradable/bioerodible polymers such aspolygalactin, poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutam-nine) and, poly(lactic acid). Biocompatiblecarriers that may be used when formulating a controlled releaseparenteral formulation are carbohydrates (e.g., dextrans), proteins(e.g., albumin), lipoproteins, or antibodies. Materials for use inimplants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(lactic acid),poly(glycolic acid) or poly(ortho esters) or combinations thereof).

Kits or Pharmaceutical Systems

The present compositions may be assembled into kits or pharmaceuticalsystems for use in ameliorating a neoplasia. Kits or pharmaceuticalsystems according to this aspect of the invention comprise a carriermeans, such as a box, carton, tube or the like, having in closeconfinement therein one or more container means, such as vials, tubes,ampoules, or bottles. The kits or pharmaceutical systems of theinvention may also comprise associated instructions for using the agentsof the invention.

Screen for Identifying Agents

In certain embodiments, the methods described herein can be used toidentify agents that can complement treatment with STAT3 inhibitors orcan identify more specific or targeted agents. For example, althoughJSI-124 may show potential antitumor effects through inhibition ofSTAT3, other off-target proinflammatory pathways are activated,emphasizing that more careful and thorough preclinical investigationsmust be implemented to prevent potential harmful effects (see, e.g.,McFarland et al., Activation of the NF-κB pathway by the STAT3 inhibitorJSI-124 in human glioblastoma cells, Mol Cancer Res. 2013 May;11(5):494-505). Thus, agents that provide a comparable or enhancedtherapeutic effect can be identified. Agents providing less side effectsmay also be identified. In certain embodiments, the agents areidentified from a small molecule library as known in the art.

In certain embodiments, screening methods employ microfluidic devices,such as described in WO 2017/075549, “High-Throughput Dynamic ReagentDelivery System.” For example, the microfluidic device can be used toestablish a gradient of one compound (e.g., JSI-124) and anothercompound can be tested for synergistic effects. Such an assay may beused to identify combination treatments that require lower doses andthus less potential side effects.

In certain embodiments, one or more agents targeting one or more genesas described herein are used in a combination treatment (e.g., STAT3inhibitor and another agent targeting a gene as described herein).

In certain embodiments, screening methods employ PDX mouse models asdescribed herein. In certain embodiments, PDX models can be treated witha chemotherapeutic agent (e.g., platinum based therapy). Agents can bescreened for the ability to sensitize or over-come platinum resistance.In certain embodiments, tumor cells grown in culture can be screened asdescribed herein for JSI-124. In certain embodiments 2D or 3D culturesare screened. In certain embodiments, OVACR4, OVACR8, OVASHO or TYKNUcells are screened. In certain embodiments, combination therapies arescreened. In certain embodiments, ex vivo cultures of platinum resistantpatients are screened. In certain embodiments, reporter cell linesexpressing a reporter (e.g., luciferase) are used to screen forcompounds capable of modulating a gene signature as described herein.The reporter can be specific for a gene as described herein. One or morereporters for one or more genes may also be used. In certainembodiments, spheroid cultures are screened.

In certain embodiments, methods of screening utilize next generationsequencing. In certain embodiments, single cell sequencing is performed.In certain embodiments, the invention involves plate based single cellRNA sequencing (see, e.g., Picelli, S. et al., 2014, “Full-lengthRNA-seq from single cells using Smart-seq2” Nature protocols 9, 171-181,doi:10.1038/nprot.2014.006).

In certain embodiments, the invention involves high-throughputsingle-cell RNA-seq and/or targeted nucleic acid profiling (for example,sequencing, quantitative reverse transcription polymerase chainreaction, and the like) where the RNAs from different cells are taggedindividually, allowing a single library to be created while retainingthe cell identity of each read. In this regard reference is made toMacosko et al., 2015, “Highly Parallel Genome-wide Expression Profilingof Individual Cells Using Nanoliter Droplets” Cell 161, 1202-1214;International patent application number PCT/US2015/049178, published asWO2016/040476 on Mar. 17, 2016; Klein et al., 2015, “Droplet Barcodingfor Single-Cell Transcriptomics Applied to Embryonic Stem Cells” Cell161, 1187-1201; International patent application numberPCT/US2016/027734, published as WO2016168584A1 on Oct. 20, 2016; Zheng,et al., 2016, “Haplotyping germline and cancer genomes withhigh-throughput linked-read sequencing” Nature Biotechnology 34,303-311; Zheng, et al., 2017, “Massively parallel digitaltranscriptional profiling of single cells” Nat. Commun. 8, 14049 doi:10.1038/ncomms14049; International patent publication numberWO2014210353A2; Zilionis, et al., 2017, “Single-cell barcoding andsequencing using droplet microfluidics” Nat Protoc. January;12(1):44-73; Cao et al., 2017, “Comprehensive single celltranscriptional profiling of a multicellular organism by combinatorialindexing” bioRxiv preprint first posted online Feb. 2, 2017, doi:dx.doi.org/10.1101/104844; Rosenberg et al., 2017, “Scaling single celltranscriptomics through split pool barcoding” bioRxiv preprint firstposted online Feb. 2, 2017, doi: dx.doi.org/10.1101/105163; Vitak, etal., “Sequencing thousands of single-cell genomes with combinatorialindexing” Nature Methods, 14(3):302-308, 2017; Cao, et al.,Comprehensive single-cell transcriptional profiling of a multicellularorganism. Science, 357(6352):661-667, 2017; and Gierahn et al.,“Seq-Well: portable, low-cost RNA sequencing of single cells at highthroughput” Nature Methods 14, 395-398 (2017), all the contents anddisclosure of each of which are herein incorporated by reference intheir entirety.

In certain embodiments, the invention involves single nucleus RNAsequencing. In this regard reference is made to Swiech et al., 2014, “Invivo interrogation of gene function in the mammalian brain usingCRISPR-Cas9” Nature Biotechnology Vol. 33, pp. 102-106; Habib et al.,2016, “Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adultnewborn neurons” Science, Vol. 353, Issue 6302, pp. 925-928; Habib etal., 2017, “Massively parallel single-nucleus RNA-seq with DroNc-seq”Nat Methods. 2017 October; 14(10):955-958; and International patentapplication number PCT/US2016/059239, published as WO2017164936 on Sep.28, 2017, which are herein incorporated by reference in their entirety.

In certain embodiments, the agents according to the present inventionare screened in a perturbation assay. For example, a perturbationlibrary is introduced to a population of cells followed by treatmentwith one or more test agents (e.g., JSI-124). Genes can be identifiedthat when perturbed cause an increase or decrease in efficacy of the oneor more agents. Methods and tools for genome-scale screening ofperturbations in single cells using CRISPR-Cas9 have been described,herein referred to as perturb-seq (see e.g., Dixit et al., “Perturb-Seq:Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling ofPooled Genetic Screens” 2016, Cell 167, 1853-1866; Adamson et al., “AMultiplexed Single-Cell CRISPR Screening Platform Enables SystematicDissection of the Unfolded Protein Response” 2016, Cell 167, 1867-1882;and International publication serial number WO/2017/075294). In certainembodiments, the network of genes affected by an agent may beidentified. In one embodiment, the method comprises (1) introducingsingle-order or combinatorial perturbations to a population of cells,(2) treating the cells with an agent, (3) measuring genomic, genetic,proteomic, epigenetic and/or phenotypic differences in single cells and(4) assigning a perturbation(s) to the single cells.

In certain embodiments, a CRISPR system is used to create an INDEL at atarget gene. In other embodiments, epigenetic screening is performed byapplying CRISPRa/i/x technology (see, e.g., Konermann et al.“Genome-scale transcriptional activation by an engineered CRISPR-Cas9complex” Nature. 2014 Dec. 10. doi: 10.1038/nature14136; Qi, L. S., etal. (2013). “Repurposing CRISPR as an RNA-guided platform forsequence-specific control of gene expression”. Cell. 152 (5): 1173-83;Gilbert, L. A., et al., (2013). “CRISPR-mediated modular RNA-guidedregulation of transcription in eukaryotes”. Cell. 154 (2): 442-51; Komoret al., 2016, Programmable editing of a target base in genomic DNAwithout double-stranded DNA cleavage, Nature 533, 420-424; Nishida etal., 2016, Targeted nucleotide editing using hybrid prokaryotic andvertebrate adaptive immune systems, Science 353(6305); Yang et al.,2016, Engineering and optimizing deaminase fusions for genome editing,Nat Commun. 7:13330; Hess et al., 2016, Directed evolution usingdCas9-targeted somatic hypermutation in mammalian cells, Nature Methods13, 1036-1042; and Ma et al., 2016, Targeted AID mediated mutagenesis(TAM) enables efficient genomic diversification in mammalian cells,Nature Methods 13, 1029-1035). Numerous genetic variants associated withdisease phenotypes are found to be in non-coding region of the genome,and frequently coincide with transcription factor (TF) binding sites andnon-coding RNA genes. Not being bound by a theory, CRISPRa/i/xapproaches may be used to achieve a more thorough and preciseunderstanding of the implication of epigenetic regulation. In oneembodiment, a CRISPR system may be used to activate gene transcription.A nuclease-dead RNA-guided DNA binding domain, dCas9, tethered totranscriptional repressor domains that promote epigenetic silencing(e.g., KRAB) may be used for “CRISPRi” that represses transcription. Touse dCas9 as an activator (CRISPRa), a guide RNA is engineered to carryRNA binding motifs (e.g., MS2) that recruit effector domains fused toRNA-motif binding proteins, increasing transcription. A key dendriticcell molecule, p65, may be used as a signal amplifier, but is notrequired.

In certain embodiments, an agent screened is a genetic modifying agent.In certain embodiments, the inhibitor as described herein for treatingor preventing a gynecological tumor in a subject comprises a geneticmodifying agent.

Genetic Modifying Agents

In certain embodiments, the one or more modulating agents may be agenetic modifying agent. The genetic modifying agent may comprise aCRISPR system, a zinc finger nuclease system, a TALEN, or ameganuclease.

In general, a CRISPR-Cas or CRISPR system as used in herein and indocuments, such as WO 2014/093622 (PCT/US2013/074667), referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, a tracr(trans-activating CRISPR) sequence (e.g. tracrRNA or an active partialtracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or “RNA(s)” as that term isherein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNAand transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). See, e.g, Shmakov et al. (2015) “Discovery and FunctionalCharacterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell,DOI: dx.doi.org/10.1016/j.molcel.2015.10.008.

In certain embodiments, a protospacer adjacent motif (PAM) or PAM-likemotif directs binding of the effector protein complex as disclosedherein to the target locus of interest. In some embodiments, the PAM maybe a 5′ PAM (i.e., located upstream of the 5′ end of the protospacer).In other embodiments, the PAM may be a 3′ PAM (i.e., located downstreamof the 5′ end of the protospacer). The term “PAM” may be usedinterchangeably with the term “PFS” or “protospacer flanking site” or“protospacer flanking sequence”.

In a preferred embodiment, the CRISPR effector protein may recognize a3′ PAM. In certain embodiments, the CRISPR effector protein mayrecognize a 3′ PAM which is 5′H, wherein H is A, C or U.

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to havecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise RNA polynucleotides. The term “target RNA” refersto a RNA polynucleotide being or comprising the target sequence. Inother words, the target RNA may be a RNA polynucleotide or a part of aRNA polynucleotide to which a part of the gRNA, i.e. the guide sequence,is designed to have complementarity and to which the effector functionmediated by the complex comprising CRISPR effector protein and a gRNA isto be directed. In some embodiments, a target sequence is located in thenucleus or cytoplasm of a cell.

In certain example embodiments, the CRISPR effector protein may bedelivered using a nucleic acid molecule encoding the CRISPR effectorprotein. The nucleic acid molecule encoding a CRISPR effector protein,may advantageously be a codon optimized CRISPR effector protein. Anexample of a codon optimized sequence, is in this instance a sequenceoptimized for expression in eukaryote, e.g., humans (i.e. beingoptimized for expression in humans), or for another eukaryote, animal ormammal as herein discussed; see, e.g., SaCas9 human codon optimizedsequence in WO 2014/093622 (PCT/US2013/074667). Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species other than human, or for codonoptimization for specific organs is known. In some embodiments, anenzyme coding sequence encoding a CRISPR effector protein is a codonoptimized for expression in particular cells, such as eukaryotic cells.The eukaryotic cells may be those of or derived from a particularorganism, such as a plant or a mammal, including but not limited tohuman, or non-human eukaryote or animal or mammal as herein discussed,e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal orprimate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded. In general,codon optimization refers to a process of modifying a nucleic acidsequence for enhanced expression in the host cells of interest byreplacing at least one codon (e.g. about or more than about 1, 2, 3, 4,5, 10, 15, 20, 25, 50, or more codons) of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat host cell while maintaining the native amino acid sequence. Variousspecies exhibit particular bias for certain codons of a particular aminoacid. Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database” available at kazusa.orjp/codon/ and these tables can beadapted in a number of ways. See Nakamura, Y., et al. “Codon usagetabulated from the international DNA sequence databases: status for theyear 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codonoptimizing a particular sequence for expression in a particular hostcell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), arealso available. In some embodiments, one or more codons (e.g. 1, 2, 3,4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encodinga Cas correspond to the most frequently used codon for a particularamino acid.

In certain embodiments, the methods as described herein may compriseproviding a Cas transgenic cell in which one or more nucleic acidsencoding one or more guide RNAs are provided or introduced operablyconnected in the cell with a regulatory element comprising a promoter ofone or more gene of interest. As used herein, the term “Cas transgeniccell” refers to a cell, such as a eukaryotic cell, in which a Cas genehas been genomically integrated. The nature, type, or origin of the cellare not particularly limiting according to the present invention. Alsothe way the Cas transgene is introduced in the cell may vary and can beany method as is known in the art. In certain embodiments, the Castransgenic cell is obtained by introducing the Cas transgene in anisolated cell. In certain other embodiments, the Cas transgenic cell isobtained by isolating cells from a Cas transgenic organism. By means ofexample, and without limitation, the Cas transgenic cell as referred toherein may be derived from a Cas transgenic eukaryote, such as a Casknock-in eukaryote. Reference is made to WO 2014/093622(PCT/US13/74667), incorporated herein by reference. Methods of US PatentPublication Nos. 20120017290 and 20110265198 assigned to SangamoBioSciences, Inc. directed to targeting the Rosa locus may be modifiedto utilize the CRISPR Cas system of the present invention. Methods of USPatent Publication No. 20130236946 assigned to Cellectis directed totargeting the Rosa locus may also be modified to utilize the CRISPR Cassystem of the present invention. By means of further example referenceis made to Platt et. al. (Cell; 159(2):440-455 (2014)), describing aCas9 knock-in mouse, which is incorporated herein by reference. The Castransgene can further comprise a Lox-Stop-polyA-Lox(LSL) cassettethereby rendering Cas expression inducible by Cre recombinase.Alternatively, the Cas transgenic cell may be obtained by introducingthe Cas transgene in an isolated cell. Delivery systems for transgenesare well known in the art. By means of example, the Cas transgene may bedelivered in for instance eukaryotic cell by means of vector (e.g., AAV,adenovirus, lentivirus) and/or particle and/or nanoparticle delivery, asalso described herein elsewhere.

It will be understood by the skilled person that the cell, such as theCas transgenic cell, as referred to herein may comprise further genomicalterations besides having an integrated Cas gene or the mutationsarising from the sequence specific action of Cas when complexed with RNAcapable of guiding Cas to a target locus.

In certain aspects the invention involves vectors, e.g. for deliveringor introducing in a cell Cas and/or RNA capable of guiding Cas to atarget locus (i.e. guide RNA), but also for propagating these components(e.g. in prokaryotic cells). A used herein, a “vector” is a tool thatallows or facilitates the transfer of an entity from one environment toanother. It is a replicon, such as a plasmid, phage, or cosmid, intowhich another DNA segment may be inserted so as to bring about thereplication of the inserted segment. Generally, a vector is capable ofreplication when associated with the proper control elements. Ingeneral, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Vectorsinclude, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that comprise one or more free ends, no free ends (e.g.circular); nucleic acid molecules that comprise DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses (AAVs)). Viral vectors also includepolynucleotides carried by a virus for transfection into a host cell.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g. bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively-linked. Such vectors are referred to herein as “expressionvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety. Thus, the embodiments disclosed herein mayalso comprise transgenic cells comprising the CRISPR effector system. Incertain example embodiments, the transgenic cell may function as anindividual discrete volume. In other words samples comprising a maskingconstruct may be delivered to a cell, for example in a suitable deliveryvesicle and if the target is present in the delivery vesicle the CRISPReffector is activated and a detectable signal generated.

The vector(s) can include the regulatory element(s), e.g., promoter(s).The vector(s) can comprise Cas encoding sequences, and/or a single, butpossibly also can comprise at least 3 or 8 or 16 or 32 or 48 or 50 guideRNA(s) (e.g., sgRNAs) encoding sequences, such as 1-2, 1-3, 1-4 1-5,3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s)(e.g., sgRNAs). In a single vector there can be a promoter for each RNA(e.g., sgRNA), advantageously when there are up to about 16 RNA(s); and,when a single vector provides for more than 16 RNA(s), one or morepromoter(s) can drive expression of more than one of the RNA(s), e.g.,when there are 32 RNA(s), each promoter can drive expression of twoRNA(s), and when there are 48 RNA(s), each promoter can drive expressionof three RNA(s). By simple arithmetic and well established cloningprotocols and the teachings in this disclosure one skilled in the artcan readily practice the invention as to the RNA(s) for a suitableexemplary vector such as AAV, and a suitable promoter such as the U6promoter. For example, the packaging limit of AAV is ˜4.7 kb. The lengthof a single U6-gRNA (plus restriction sites for cloning) is 361 bp.Therefore, the skilled person can readily fit about 12-16, e.g., 13U6-gRNA cassettes in a single vector. This can be assembled by anysuitable means, such as a golden gate strategy used for TALE assembly(genome-engineering.org/taleffectors/). The skilled person can also usea tandem guide strategy to increase the number of U6-gRNAs byapproximately 1.5 times, e.g., to increase from 12-16, e.g., 13 toapproximately 18-24, e.g., about 19 U6-gRNAs. Therefore, one skilled inthe art can readily reach approximately 18-24, e.g., about 19promoter-RNAs, e.g., U6-gRNAs in a single vector, e.g., an AAV vector. Afurther means for increasing the number of promoters and RNAs in avector is to use a single promoter (e.g., U6) to express an array ofRNAs separated by cleavable sequences. And an even further means forincreasing the number of promoter-RNAs in a vector, is to express anarray of promoter-RNAs separated by cleavable sequences in the intron ofa coding sequence or gene; and, in this instance it is advantageous touse a polymerase II promoter, which can have increased expression andenable the transcription of long RNA in a tissue specific manner. (see,e.g., nar.oxfordjournals.org/content/34/7/e53. short andnature.com/mt/journal/v16/n9/abs/mt2008144a.html). In an advantageousembodiment, AAV may package U6 tandem gRNA targeting up to about 50genes. Accordingly, from the knowledge in the art and the teachings inthis disclosure the skilled person can readily make and use vector(s),e.g., a single vector, expressing multiple RNAs or guides under thecontrol or operatively or functionally linked to one or morepromoters-especially as to the numbers of RNAs or guides discussedherein, without any undue experimentation.

The guide RNA(s) encoding sequences and/or Cas encoding sequences, canbe functionally or operatively linked to regulatory element(s) and hencethe regulatory element(s) drive expression. The promoter(s) can beconstitutive promoter(s) and/or conditional promoter(s) and/or induciblepromoter(s) and/or tissue specific promoter(s). The promoter can beselected from the group consisting of RNA polymerases, pol I, pol II,pol III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter,the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolatereductase promoter, the β-actin promoter, the phosphoglycerol kinase(PGK) promoter, and the EF1α promoter. An advantageous promoter is thepromoter is U6.

Additional effectors for use according to the invention can beidentified by their proximity to cas1 genes, for example, though notlimited to, within the region 20 kb from the start of the cas1 gene and20 kb from the end of the cas1 gene. In certain embodiments, theeffector protein comprises at least one HEPN domain and at least 500amino acids, and wherein the C2c2 effector protein is naturally presentin a prokaryotic genome within 20 kb upstream or downstream of a Casgene or a CRISPR array. Non-limiting examples of Cas proteins includeCas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also knownas Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2,Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6,Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15,Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versionsthereof. In certain example embodiments, the C2c2 effector protein isnaturally present in a prokaryotic genome within 20 kb upstream ordownstream of a Cas 1 gene. The terms “orthologue” (also referred to as“ortholog” herein) and “homologue” (also referred to as “homolog”herein) are well known in the art. By means of further guidance, a“homologue” of a protein as used herein is a protein of the same specieswhich performs the same or a similar function as the protein it is ahomologue of. Homologous proteins may but need not be structurallyrelated, or are only partially structurally related. An “orthologue” ofa protein as used herein is a protein of a different species whichperforms the same or a similar function as the protein it is anorthologue of. Orthologous proteins may but need not be structurallyrelated, or are only partially structurally related.

Guide Molecules

The methods described herein may be used to screen inhibition of CRISPRsystems employing different types of guide molecules. As used herein,the term “guide sequence” and “guide molecule” in the context of aCRISPR-Cas system, comprises any polynucleotide sequence havingsufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of a nucleic acid-targeting complex to thetarget nucleic acid sequence. The guide sequences made using the methodsdisclosed herein may be a full-length guide sequence, a truncated guidesequence, a full-length sgRNA sequence, a truncated sgRNA sequence, oran E+F sgRNA sequence. In some embodiments, the degree ofcomplementarity of the guide sequence to a given target sequence, whenoptimally aligned using a suitable alignment algorithm, is about or morethan about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Incertain example embodiments, the guide molecule comprises a guidesequence that may be designed to have at least one mismatch with thetarget sequence, such that a RNA duplex formed between the guidesequence and the target sequence. Accordingly, the degree ofcomplementarity is preferably less than 99%. For instance, where theguide sequence consists of 24 nucleotides, the degree of complementarityis more particularly about 96% or less. In particular embodiments, theguide sequence is designed to have a stretch of two or more adjacentmismatching nucleotides, such that the degree of complementarity overthe entire guide sequence is further reduced. For instance, where theguide sequence consists of 24 nucleotides, the degree of complementarityis more particularly about 96% or less, more particularly, about 92% orless, more particularly about 88% or less, more particularly about 84%or less, more particularly about 80% or less, more particularly about76% or less, more particularly about 72% or less, depending on whetherthe stretch of two or more mismatching nucleotides encompasses 2, 3, 4,5, 6 or 7 nucleotides, etc. In some embodiments, aside from the stretchof one or more mismatching nucleotides, the degree of complementarity,when optimally aligned using a suitable alignment algorithm, is about ormore than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.Optimal alignment may be determined with the use of any suitablealgorithm for aligning sequences, non-limiting example of which includethe Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g., the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies;available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.),SOAP (available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). The ability of a guide sequence (within a nucleicacid-targeting guide RNA) to direct sequence-specific binding of anucleic acid-targeting complex to a target nucleic acid sequence may beassessed by any suitable assay. For example, the components of a nucleicacid-targeting CRISPR system sufficient to form a nucleic acid-targetingcomplex, including the guide sequence to be tested, may be provided to ahost cell having the corresponding target nucleic acid sequence, such asby transfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget nucleic acid sequence (or a sequence in the vicinity thereof) maybe evaluated in a test tube by providing the target nucleic acidsequence, components of a nucleic acid-targeting complex, including theguide sequence to be tested and a control guide sequence different fromthe test guide sequence, and comparing binding or rate of cleavage at orin the vicinity of the target sequence between the test and controlguide sequence reactions. Other assays are possible, and will occur tothose skilled in the art. A guide sequence, and hence a nucleicacid-targeting guide RNA may be selected to target any target nucleicacid sequence.

In certain embodiments, the guide sequence or spacer length of the guidemolecules is from 15 to 50 nt. In certain embodiments, the spacer lengthof the guide RNA is at least 15 nucleotides. In certain embodiments, thespacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23,or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt,e.g., 24, 25, 26, or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt,from 30-35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.In certain example embodiment, the guide sequence is 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39 40, 41, 42, 43, 44, 45, 46, 47 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100 nt.

In some embodiments, the guide sequence is an RNA sequence of between 10to 50 nt in length, but more particularly of about 20-30 ntadvantageously about 20 nt, 23-25 nt or 24 nt. The guide sequence isselected so as to ensure that it hybridizes to the target sequence. Thisis described more in detail below. Selection can encompass further stepswhich increase efficacy and specificity.

In some embodiments, the guide sequence has a canonical length (e.g.,about 15-30 nt) is used to hybridize with the target RNA or DNA. In someembodiments, a guide molecule is longer than the canonical length(e.g., >30 nt) is used to hybridize with the target RNA or DNA, suchthat a region of the guide sequence hybridizes with a region of the RNAor DNA strand outside of the Cas-guide target complex. This can be ofinterest where additional modifications, such deamination of nucleotidesis of interest. In alternative embodiments, it is of interest tomaintain the limitation of the canonical guide sequence length.

In some embodiments, the sequence of the guide molecule (direct repeatand/or spacer) is selected to reduce the degree secondary structurewithin the guide molecule. In some embodiments, about or less than about75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of thenucleotides of the nucleic acid-targeting guide RNA participate inself-complementary base pairing when optimally folded. Optimal foldingmay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g., A.R. Gruber et al., 2008, Cell106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology27(12): 1151-62).

In some embodiments, it is of interest to reduce the susceptibility ofthe guide molecule to RNA cleavage, such as to cleavage by Cas13.Accordingly, in particular embodiments, the guide molecule is adjustedto avoid cleavage by Cas13 or other RNA-cleaving enzymes.

In certain embodiments, the guide molecule comprises non-naturallyoccurring nucleic acids and/or non-naturally occurring nucleotidesand/or nucleotide analogs, and/or chemically modifications. Preferably,these non-naturally occurring nucleic acids and non-naturally occurringnucleotides are located outside the guide sequence. Non-naturallyoccurring nucleic acids can include, for example, mixtures of naturallyand non-naturally occurring nucleotides. Non-naturally occurringnucleotides and/or nucleotide analogs may be modified at the ribose,phosphate, and/or base moiety. In an embodiment of the invention, aguide nucleic acid comprises ribonucleotides and non-ribonucleotides. Inone such embodiment, a guide comprises one or more ribonucleotides andone or more deoxyribonucleotides. In an embodiment of the invention, theguide comprises one or more non-naturally occurring nucleotide ornucleotide analog such as a nucleotide with phosphorothioate linkage, alocked nucleic acid (LNA) nucleotides comprising a methylene bridgebetween the 2′ and 4′ carbons of the ribose ring, or bridged nucleicacids (BNA). Other examples of modified nucleotides include 2′-O-methylanalogs, 2′-deoxy analogs, or 2′-fluoro analogs. Further examples ofmodified bases include, but are not limited to, 2-aminopurine,5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. Examples ofguide RNA chemical modifications include, without limitation,incorporation of 2′-O-methyl (M), 2′-O-methyl 3′ phosphorothioate (MS),S-constrained ethyl(cEt), or 2′-O-methyl 3′ thioPACE (MSP) at one ormore terminal nucleotides. Such chemically modified guides can compriseincreased stability and increased activity as compared to unmodifiedguides, though on-target vs. off-target specificity is not predictable.(See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290,published online 29 Jun. 2015 Ragdarm et al., 0215, PNAS, E7110-E7111;Allerson et al., J Med. Chem. 2005, 48:901-904; Bramsen et al., Front.Genet., 2012, 3:154; Deng et al., PNAS, 2015, 112:11870-11875; Sharma etal., MedChemComm., 2014, 5:1454-1471; Hendel et al., Nat. Biotechnol.(2015) 33(9): 985-989; Li et al., Nature Biomedical Engineering, 2017,1, 0066 DOI:10.1038/s41551-017-0066). In some embodiments, the 5′ and/or3′ end of a guide RNA is modified by a variety of functional moietiesincluding fluorescent dyes, polyethylene glycol, cholesterol, proteins,or detection tags. (See Kelly et al., 2016, J. Biotech. 233:74-83). Incertain embodiments, a guide comprises ribonucleotides in a region thatbinds to a target RNA and one or more deoxyribonucletides and/ornucleotide analogs in a region that binds to Cas13. In an embodiment ofthe invention, deoxyribonucleotides and/or nucleotide analogs areincorporated in engineered guide structures, such as, withoutlimitation, stem-loop regions, and the seed region. For Cas13 guide, incertain embodiments, the modification is not in the 5′-handle of thestem-loop regions. Chemical modification in the 5′-handle of thestem-loop region of a guide may abolish its function (see Li, et al.,Nature Biomedical Engineering, 2017, 1:0066). In certain embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75nucleotides of a guide is chemically modified. In some embodiments, 3-5nucleotides at either the 3′ or the 5′ end of a guide is chemicallymodified. In some embodiments, only minor modifications are introducedin the seed region, such as 2′-F modifications. In some embodiments,2′-F modification is introduced at the 3′ end of a guide. In certainembodiments, three to five nucleotides at the 5′ and/or the 3′ end ofthe guide are chemicially modified with 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), S-constrained ethyl(cEt), or 2′-O-methyl 3′thioPACE (MSP). Such modification can enhance genome editing efficiency(see Hendel et al., Nat. Biotechnol. (2015) 33(9): 985-989). In certainembodiments, all of the phosphodiester bonds of a guide are substitutedwith phosphorothioates (PS) for enhancing levels of gene disruption. Incertain embodiments, more than five nucleotides at the 5′ and/or the 3′end of the guide are chemicially modified with 2′-O-Me, 2′-F orS-constrained ethyl(cEt). Such chemically modified guide can mediateenhanced levels of gene disruption (see Ragdarm et al., 0215, PNAS,E7110-E7111). In an embodiment of the invention, a guide is modified tocomprise a chemical moiety at its 3′ and/or 5′ end. Such moietiesinclude, but are not limited to amine, azide, alkyne, thio,dibenzocyclooctyne (DBCO), or Rhodamine. In certain embodiment, thechemical moiety is conjugated to the guide by a linker, such as an alkylchain. In certain embodiments, the chemical moiety of the modified guidecan be used to attach the guide to another molecule, such as DNA, RNA,protein, or nanoparticles. Such chemically modified guide can be used toidentify or enrich cells generically edited by a CRISPR system (see Leeet al., eLife, 2017, 6:e25312, DOI:10.7554).

In some embodiments, the modification to the guide is a chemicalmodification, an insertion, a deletion or a split. In some embodiments,the chemical modification includes, but is not limited to, incorporationof 2′-O-methyl (M) analogs, 2′-deoxy analogs, 2-thiouridine analogs,N6-methyladenosine analogs, 2′-fluoro analogs, 2-aminopurine,5-bromo-uridine, pseudouridine (Ψ), N1-methylpseudouridine (me1Ψ),5-methoxyuridine(5moU), inosine, 7-methylguanosine, 2′-O-methyl3′phosphorothioate (MS), S-constrained ethyl(cEt), phosphorothioate(PS), or 2′-O-methyl 3′thioPACE (MSP). In some embodiments, the guidecomprises one or more of phosphorothioate modifications. In certainembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or 25 nucleotides of the guide are chemicallymodified. In certain embodiments, one or more nucleotides in the seedregion are chemically modified. In certain embodiments, one or morenucleotides in the 3′-terminus are chemically modified. In certainembodiments, none of the nucleotides in the 5′-handle is chemicallymodified. In some embodiments, the chemical modification in the seedregion is a minor modification, such as incorporation of a 2′-fluoroanalog. In a specific embodiment, one nucleotide of the seed region isreplaced with a 2′-fluoro analog. In some embodiments, 5 to 10nucleotides in the 3′-terminus are chemically modified. Such chemicalmodifications at the 3′-terminus of the Cas13 CrRNA may improve Cas13activity. In a specific embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10nucleotides in the 3′-terminus are replaced with 2′-fluoro analogues. Ina specific embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides inthe 3′-terminus are replaced with 2′-O-methyl (M) analogs.

In some embodiments, the loop of the 5′-handle of the guide is modified.In some embodiments, the loop of the 5′-handle of the guide is modifiedto have a deletion, an insertion, a split, or chemical modifications. Incertain embodiments, the modified loop comprises 3, 4, or 5 nucleotides.In certain embodiments, the loop comprises the sequence of UCUU, UUUU,UAUU, or UGUU (SEQ. I.D. Nos. 1-4).

In some embodiments, the guide molecule forms a stemloop with a separatenon-covalently linked sequence, which can be DNA or RNA. In particularembodiments, the sequences forming the guide are first synthesized usingthe standard phosphoramidite synthetic protocol (Herdewijn, P., ed.,Methods in Molecular Biology Col 288, Oligonucleotide Synthesis: Methodsand Applications, Humana Press, New Jersey (2012)). In some embodiments,these sequences can be functionalized to contain an appropriatefunctional group for ligation using the standard protocol known in theart (Hermanson, G. T., Bioconjugate Techniques, Academic Press (2013)).Examples of functional groups include, but are not limited to, hydroxyl,amine, carboxylic acid, carboxylic acid halide, carboxylic acid activeester, aldehyde, carbonyl, chlorocarbonyl, imidazolylcarbonyl,hydrozide, semicarbazide, thio semicarbazide, thiol, maleimide,haloalkyl, sufonyl, ally, propargyl, diene, alkyne, and azide. Once thissequence is functionalized, a covalent chemical bond or linkage can beformed between this sequence and the direct repeat sequence. Examples ofchemical bonds include, but are not limited to, those based oncarbamates, ethers, esters, amides, imines, amidines, aminotrizines,hydrozone, disulfides, thioethers, thioesters, phosphorothioates,phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides,ureas, thioureas, hydrazide, oxime, triazole, photolabile linkages, C—Cbond forming groups such as Diels-Alder cyclo-addition pairs orring-closing metathesis pairs, and Michael reaction pairs.

In some embodiments, these stem-loop forming sequences can be chemicallysynthesized. In some embodiments, the chemical synthesis uses automated,solid-phase oligonucleotide synthesis machines with 2′-acetoxyethylorthoester (2′-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317: 3-18) or2′-thionocarbamate (2′-TC) chemistry (Dellinger et al., J. Am. Chem.Soc. (2011) 133: 11540-11546; Hendel et al., Nat. Biotechnol. (2015)33:985-989).

In certain embodiments, the guide molecule comprises (1) a guidesequence capable of hybridizing to a target locus and (2) a tracr mateor direct repeat sequence whereby the direct repeat sequence is locatedupstream (i.e., 5′) from the guide sequence. In a particular embodimentthe seed sequence (i.e. the sequence essential critical for recognitionand/or hybridization to the sequence at the target locus) of th guidesequence is approximately within the first 10 nucleotides of the guidesequence.

In a particular embodiment the guide molecule comprises a guide sequencelinked to a direct repeat sequence, wherein the direct repeat sequencecomprises one or more stem loops or optimized secondary structures. Inparticular embodiments, the direct repeat has a minimum length of 16 ntsand a single stem loop. In further embodiments the direct repeat has alength longer than 16 nts, preferably more than 17 nts, and has morethan one stem loops or optimized secondary structures. In particularembodiments the guide molecule comprises or consists of the guidesequence linked to all or part of the natural direct repeat sequence. Atypical Type V or Type VI CRISPR-cas guide molecule comprises (in 3′ to5′ direction or in 5′ to 3′ direction): a guide sequence a firstcomplimentary stretch (the “repeat”), a loop (which is typically 4 or 5nucleotides long), a second complimentary stretch (the “anti-repeat”being complimentary to the repeat), and a poly A (often poly U in RNA)tail (terminator). In certain embodiments, the direct repeat sequenceretains its natural architecture and forms a single stem loop. Inparticular embodiments, certain aspects of the guide architecture can bemodified, for example by addition, subtraction, or substitution offeatures, whereas certain other aspects of guide architecture aremaintained. Preferred locations for engineered guide moleculemodifications, including but not limited to insertions, deletions, andsubstitutions include guide termini and regions of the guide moleculethat are exposed when complexed with the CRISPR-Cas protein and/ortarget, for example the stemloop of the direct repeat sequence.

In particular embodiments, the stem comprises at least about 4 bpcomprising complementary X and Y sequences, although stems of more,e.g., 5, 6, 7, 8, 9, 10, 11 or 12 or fewer, e.g., 3, 2, base pairs arealso contemplated. Thus, for example X2-10 and Y2-10 (wherein X and Yrepresent any complementary set of nucleotides) may be contemplated. Inone aspect, the stem made of the X and Y nucleotides, together with theloop will form a complete hairpin in the overall secondary structure;and, this may be advantageous and the amount of base pairs can be anyamount that forms a complete hairpin. In one aspect, any complementaryX:Y basepairing sequence (e.g., as to length) is tolerated, so long asthe secondary structure of the entire guide molecule is preserved. Inone aspect, the loop that connects the stem made of X:Y basepairs can beany sequence of the same length (e.g., 4 or 5 nucleotides) or longerthat does not interrupt the overall secondary structure of the guidemolecule. In one aspect, the stemloop can further comprise, e.g. an MS2aptamer. In one aspect, the stem comprises about 5-7 bp comprisingcomplementary X and Y sequences, although stems of more or fewerbasepairs are also contemplated. In one aspect, non-Watson Crickbasepairing is contemplated, where such pairing otherwise generallypreserves the architecture of the stemloop at that position.

In particular embodiments the natural hairpin or stemloop structure ofthe guide molecule is extended or replaced by an extended stemloop. Ithas been demonstrated that extension of the stem can enhance theassembly of the guide molecule with the CRISPR-Cas protein (Chen et al.Cell. (2013); 155(7): 1479-1491). In particular embodiments the stem ofthe stemloop is extended by at least 1, 2, 3, 4, 5 or more complementarybasepairs (i.e. corresponding to the addition of 2, 4, 6, 8, 10 or morenucleotides in the guide molecule). In particular embodiments these arelocated at the end of the stem, adjacent to the loop of the stemloop.

In particular embodiments, the susceptibility of the guide molecule toRNAses or to decreased expression can be reduced by slight modificationsof the sequence of the guide molecule which do not affect its function.For instance, in particular embodiments, premature termination oftranscription, such as premature transcription of U6 Pol-III, can beremoved by modifying a putative Pol-III terminator (4 consecutive U's)in the guide molecules sequence. Where such sequence modification isrequired in the stemloop of the guide molecule, it is preferably ensuredby a basepair flip.

In a particular embodiment the direct repeat may be modified to compriseone or more protein-binding RNA aptamers. In a particular embodiment,one or more aptamers may be included such as part of optimized secondarystructure. Such aptamers may be capable of binding a bacteriophage coatprotein as detailed further herein.

In some embodiments, the guide molecule forms a duplex with a target RNAcomprising at least one target cytosine residue to be edited. Uponhybridization of the guide RNA molecule to the target RNA, the cytidinedeaminase binds to the single strand RNA in the duplex made accessibleby the mismatch in the guide sequence and catalyzes deamination of oneor more target cytosine residues comprised within the stretch ofmismatching nucleotides.

A guide sequence, and hence a nucleic acid-targeting guide RNA may beselected to target any target nucleic acid sequence. The target sequencemay be mRNA.

In certain embodiments, the target sequence should be associated with aPAM (protospacer adjacent motif) or PFS (protospacer flanking sequenceor site); that is, a short sequence recognized by the CRISPR complex.Depending on the nature of the CRISPR-Cas protein, the target sequenceshould be selected such that its complementary sequence in the DNAduplex (also referred to herein as the non-target sequence) is upstreamor downstream of the PAM. In the embodiments of the present inventionwhere the CRISPR-Cas protein is a Cas13 protein, the complementarysequence of the target sequence is downstream or 3′ of the PAM orupstream or 5′ of the PAM. The precise sequence and length requirementsfor the PAM differ depending on the Cas13 protein used, but PAMs aretypically 2-5 base pair sequences adjacent the protospacer (that is, thetarget sequence). Examples of the natural PAM sequences for differentCas13 orthologues are provided herein below and the skilled person willbe able to identify further PAM sequences for use with a given Cas13protein.

Further, engineering of the PAM Interacting (PI) domain may allowprograming of PAM specificity, improve target site recognition fidelity,and increase the versatility of the CRISPR-Cas protein, for example asdescribed for Cas9 in Kleinstiver B P et al. Engineered CRISPR-Cas9nucleases with altered PAM specificities. Nature. 2015 Jul. 23;523(7561):481-5. doi: 10.1038/nature14592. As further detailed herein,the skilled person will understand that Cas13 proteins may be modifiedanalogously.

In particular embodiment, the guide is an escorted guide. By “escorted”is meant that the CRISPR-Cas system or complex or guide is delivered toa selected time or place within a cell, so that activity of theCRISPR-Cas system or complex or guide is spatially or temporallycontrolled. For example, the activity and destination of the 3CRISPR-Cas system or complex or guide may be controlled by an escort RNAaptamer sequence that has binding affinity for an aptamer ligand, suchas a cell surface protein or other localized cellular component.Alternatively, the escort aptamer may for example be responsive to anaptamer effector on or in the cell, such as a transient effector, suchas an external energy source that is applied to the cell at a particulartime.

The escorted CRISPR-Cas systems or complexes have a guide molecule witha functional structure designed to improve guide molecule structure,architecture, stability, genetic expression, or any combination thereof.Such a structure can include an aptamer.

Aptamers are biomolecules that can be designed or selected to bindtightly to other ligands, for example using a technique calledsystematic evolution of ligands by exponential enrichment (SELEX; TuerkC, Gold L: “Systematic evolution of ligands by exponential enrichment:RNA ligands to bacteriophage T4 DNA polymerase.” Science 1990,249:505-510). Nucleic acid aptamers can for example be selected frompools of random-sequence oligonucleotides, with high binding affinitiesand specificities for a wide range of biomedically relevant targets,suggesting a wide range of therapeutic utilities for aptamers (Keefe,Anthony D., Supriya Pai, and Andrew Ellington. “Aptamers astherapeutics.” Nature Reviews Drug Discovery 9.7 (2010): 537-550). Thesecharacteristics also suggest a wide range of uses for aptamers as drugdelivery vehicles (Levy-Nissenbaum, Etgar, et al. “Nanotechnology andaptamers: applications in drug delivery.” Trends in biotechnology 26.8(2008): 442-449; and, Hicke B J, Stephens A W. “Escort aptamers: adelivery service for diagnosis and therapy.” J Clin Invest 2000,106:923-928.). Aptamers may also be constructed that function asmolecular switches, responding to a que by changing properties, such asRNA aptamers that bind fluorophores to mimic the activity of greenflourescent protein (Paige, Jeremy S., Karen Y. Wu, and Samie R.Jaffrey. “RNA mimics of green fluorescent protein.” Science 333.6042(2011): 642-646). It has also been suggested that aptamers may be usedas components of targeted siRNA therapeutic delivery systems, forexample targeting cell surface proteins (Zhou, Jiehua, and John J.Rossi. “Aptamer-targeted cell-specific RNA interference.” Silence 1.1(2010): 4).

Accordingly, in particular embodiments, the guide molecule is modified,e.g., by one or more aptamer(s) designed to improve guide moleculedelivery, including delivery across the cellular membrane, tointracellular compartments, or into the nucleus. Such a structure caninclude, either in addition to the one or more aptamer(s) or withoutsuch one or more aptamer(s), moiety(ies) so as to render the guidemolecule deliverable, inducible or responsive to a selected effector.The invention accordingly comprehends an guide molecule that responds tonormal or pathological physiological conditions, including withoutlimitation pH, hypoxia, O₂ concentration, temperature, proteinconcentration, enzymatic concentration, lipid structure, light exposure,mechanical disruption (e.g. ultrasound waves), magnetic fields, electricfields, or electromagnetic radiation.

Light responsiveness of an inducible system may be achieved via theactivation and binding of cryptochrome-2 and CIB1. Blue lightstimulation induces an activating conformational change incryptochrome-2, resulting in recruitment of its binding partner CIB1.This binding is fast and reversible, achieving saturation in <15 secfollowing pulsed stimulation and returning to baseline <15 min after theend of stimulation. These rapid binding kinetics result in a systemtemporally bound only by the speed of transcription/translation andtranscript/protein degradation, rather than uptake and clearance ofinducing agents. Crytochrome-2 activation is also highly sensitive,allowing for the use of low light intensity stimulation and mitigatingthe risks of phototoxicity. Further, in a context such as the intactmammalian brain, variable light intensity may be used to control thesize of a stimulated region, allowing for greater precision than vectordelivery alone may offer.

The invention contemplates energy sources such as electromagneticradiation, sound energy or thermal energy to induce the guide.Advantageously, the electromagnetic radiation is a component of visiblelight. In a preferred embodiment, the light is a blue light with awavelength of about 450 to about 495 nm. In an especially preferredembodiment, the wavelength is about 488 nm. In another preferredembodiment, the light stimulation is via pulses. The light power mayrange from about 0-9 mW/cm². In a preferred embodiment, a stimulationparadigm of as low as 0.25 sec every 15 sec should result in maximalactivation.

The chemical or energy sensitive guide may undergo a conformationalchange upon induction by the binding of a chemical source or by theenergy allowing it act as a guide and have the Cas13 CRISPR-Cas systemor complex function. The invention can involve applying the chemicalsource or energy so as to have the guide function and the Cas13CRISPR-Cas system or complex function; and optionally furtherdetermining that the expression of the genomic locus is altered.

There are several different designs of this chemical induciblesystem: 1. ABI-PYL based system inducible by Abscisic Acid (ABA) (see,e.g., stke.sciencemag.org/cgi/content/abstract/sigtrans; 4/164/r52), 2.FKBP-FRB based system inducible by rapamycin (or related chemicals basedon rapamycin) (see, e.g.,www.nature.com/nmeth/journal/v2/n6/full/nmeth763.html), 3. GID1-GAIbased system inducible by Gibberellin (GA) (see, e.g.,www.nature.com/nchembio/journal/v8/n5/full/nchembio.922. html).

A chemical inducible system can be an estrogen receptor (ER) basedsystem inducible by 4-hydroxytamoxifen (4OHT) (see, e.g.,www.pnas.org/content/104/3/1027. abstract). A mutated ligand-bindingdomain of the estrogen receptor called ERT2 translocates into thenucleus of cells upon binding of 4-hydroxytamoxifen. In furtherembodiments of the invention any naturally occurring or engineeredderivative of any nuclear receptor, thyroid hormone receptor, retinoicacid receptor, estrogren receptor, estrogen-related receptor,glucocorticoid receptor, progesterone receptor, androgen receptor may beused in inducible systems analogous to the ER based inducible system.

Another inducible system is based on the design using Transient receptorpotential (TRP) ion channel based system inducible by energy, heat orradio-wave (see, e.g., www.sciencemag.org/content/336/6081/604). TheseTRP family proteins respond to different stimuli, including light andheat. When this protein is activated by light or heat, the ion channelwill open and allow the entering of ions such as calcium into the plasmamembrane. This influx of ions will bind to intracellular ion interactingpartners linked to a polypeptide including the guide and the othercomponents of the Cas13 CRISPR-Cas complex or system, and the bindingwill induce the change of sub-cellular localization of the polypeptide,leading to the entire polypeptide entering the nucleus of cells. Onceinside the nucleus, the guide protein and the other components of theCas13 CRISPR-Cas complex will be active and modulating target geneexpression in cells.

While light activation may be an advantageous embodiment, sometimes itmay be disadvantageous especially for in vivo applications in which thelight may not penetrate the skin or other organs. In this instance,other methods of energy activation are contemplated, in particular,electric field energy and/or ultrasound which have a similar effect.

Electric field energy is preferably administered substantially asdescribed in the art, using one or more electric pulses of from about 1Volt/cm to about 10 kVolts/cm under in vivo conditions. Instead of or inaddition to the pulses, the electric field may be delivered in acontinuous manner. The electric pulse may be applied for between 1 μsand 500 milliseconds, preferably between 1 μs and 100 milliseconds. Theelectric field may be applied continuously or in a pulsed manner for 5about minutes.

As used herein, ‘electric field energy’ is the electrical energy towhich a cell is exposed. Preferably the electric field has a strength offrom about 1 Volt/cm to about 10 kVolts/cm or more under in vivoconditions (see WO97/49450).

As used herein, the term “electric field” includes one or more pulses atvariable capacitance and voltage and including exponential and/or squarewave and/or modulated wave and/or modulated square wave forms.References to electric fields and electricity should be taken to includereference the presence of an electric potential difference in theenvironment of a cell. Such an environment may be set up by way ofstatic electricity, alternating current (AC), direct current (DC), etc,as known in the art. The electric field may be uniform, non-uniform orotherwise, and may vary in strength and/or direction in a time dependentmanner.

Single or multiple applications of electric field, as well as single ormultiple applications of ultrasound are also possible, in any order andin any combination. The ultrasound and/or the electric field may bedelivered as single or multiple continuous applications, or as pulses(pulsatile delivery).

Electroporation has been used in both in vitro and in vivo procedures tointroduce foreign material into living cells. With in vitroapplications, a sample of live cells is first mixed with the agent ofinterest and placed between electrodes such as parallel plates. Then,the electrodes apply an electrical field to the cell/implant mixture.Examples of systems that perform in vitro electroporation include theElectro Cell Manipulator ECM600 product, and the Electro Square PoratorT820, both made by the BTX Division of Genetronics, Inc (see U.S. Pat.No. 5,869,326).

The known electroporation techniques (both in vitro and in vivo)function by applying a brief high voltage pulse to electrodes positionedaround the treatment region. The electric field generated between theelectrodes causes the cell membranes to temporarily become porous,whereupon molecules of the agent of interest enter the cells. In knownelectroporation applications, this electric field comprises a singlesquare wave pulse on the order of 1000 V/cm, of about 100 .mu.sduration. Such a pulse may be generated, for example, in knownapplications of the Electro Square Porator T820.

Preferably, the electric field has a strength of from about 1 V/cm toabout 10 kV/cm under in vitro conditions. Thus, the electric field mayhave a strength of 1 V/cm, 2 V/cm, 3 V/cm, 4 V/cm, 5 V/cm, 6 V/cm, 7V/cm, 8 V/cm, 9 V/cm, 10 V/cm, 20 V/cm, 50 V/cm, 100 V/cm, 200 V/cm, 300V/cm, 400 V/cm, 500 V/cm, 600 V/cm, 700 V/cm, 800 V/cm, 900 V/cm, 1kV/cm, 2 kV/cm, 5 kV/cm, 10 kV/cm, 20 kV/cm, 50 kV/cm or more. Morepreferably from about 0.5 kV/cm to about 4.0 kV/cm under in vitroconditions. Preferably the electric field has a strength of from about 1V/cm to about 10 kV/cm under in vivo conditions. However, the electricfield strengths may be lowered where the number of pulses delivered tothe target site are increased. Thus, pulsatile delivery of electricfields at lower field strengths is envisaged.

Preferably the application of the electric field is in the form ofmultiple pulses such as double pulses of the same strength andcapacitance or sequential pulses of varying strength and/or capacitance.As used herein, the term “pulse” includes one or more electric pulses atvariable capacitance and voltage and including exponential and/or squarewave and/or modulated wave/square wave forms.

Preferably the electric pulse is delivered as a waveform selected froman exponential wave form, a square wave form, a modulated wave form anda modulated square wave form.

A preferred embodiment employs direct current at low voltage. Thus,Applicants disclose the use of an electric field which is applied to thecell, tissue or tissue mass at a field strength of between 1V/cm and20V/cm, for a period of 100 milliseconds or more, preferably 15 minutesor more.

Ultrasound is advantageously administered at a power level of from about0.05 W/cm2 to about 100 W/cm2. Diagnostic or therapeutic ultrasound maybe used, or combinations thereof.

As used herein, the term “ultrasound” refers to a form of energy whichconsists of mechanical vibrations the frequencies of which are so highthey are above the range of human hearing. Lower frequency limit of theultrasonic spectrum may generally be taken as about 20 kHz. Mostdiagnostic applications of ultrasound employ frequencies in the range 1and 15 MHz′ (From Ultrasonics in Clinical Diagnosis, P. N. T. Wells,ed., 2nd. Edition, Publ. Churchill Livingstone [Edinburgh, London & NY,1977]).

Ultrasound has been used in both diagnostic and therapeuticapplications. When used as a diagnostic tool (“diagnostic ultrasound”),ultrasound is typically used in an energy density range of up to about100 mW/cm2 (FDA recommendation), although energy densities of up to 750mW/cm2 have been used. In physiotherapy, ultrasound is typically used asan energy source in a range up to about 3 to 4 W/cm2 (WHOrecommendation). In other therapeutic applications, higher intensitiesof ultrasound may be employed, for example, HIFU at 100 W/cm up to 1kW/cm2 (or even higher) for short periods of time. The term “ultrasound”as used in this specification is intended to encompass diagnostic,therapeutic and focused ultrasound.

Focused ultrasound (FUS) allows thermal energy to be delivered withoutan invasive probe (see Morocz et al 1998 Journal of Magnetic ResonanceImaging Vol. 8, No. 1, pp. 136-142. Another form of focused ultrasoundis high intensity focused ultrasound (HIFU) which is reviewed byMoussatov et al in Ultrasonics (1998) Vol. 36, No. 8, pp. 893-900 andTranHuuHue et al in Acustica (1997) Vol. 83, No. 6, pp. 1103-1106.

Preferably, a combination of diagnostic ultrasound and a therapeuticultrasound is employed. This combination is not intended to be limiting,however, and the skilled reader will appreciate that any variety ofcombinations of ultrasound may be used. Additionally, the energydensity, frequency of ultrasound, and period of exposure may be varied.

Preferably the exposure to an ultrasound energy source is at a powerdensity of from about 0.05 to about 100 Wcm-2. Even more preferably, theexposure to an ultrasound energy source is at a power density of fromabout 1 to about 15 Wcm-2.

Preferably the exposure to an ultrasound energy source is at a frequencyof from about 0.015 to about 10.0 MHz. More preferably the exposure toan ultrasound energy source is at a frequency of from about 0.02 toabout 5.0 MHz or about 6.0 MHz. Most preferably, the ultrasound isapplied at a frequency of 3 MHz.

Preferably the exposure is for periods of from about 10 milliseconds toabout 60 minutes. Preferably the exposure is for periods of from about 1second to about 5 minutes. More preferably, the ultrasound is appliedfor about 2 minutes. Depending on the particular target cell to bedisrupted, however, the exposure may be for a longer duration, forexample, for 15 minutes.

Advantageously, the target tissue is exposed to an ultrasound energysource at an acoustic power density of from about 0.05 Wcm-2 to about 10Wcm-2 with a frequency ranging from about 0.015 to about 10 MHz (see WO98/52609). However, alternatives are also possible, for example,exposure to an ultrasound energy source at an acoustic power density ofabove 100 Wcm-2, but for reduced periods of time, for example, 1000Wcm-2 for periods in the millisecond range or less.

Preferably the application of the ultrasound is in the form of multiplepulses; thus, both continuous wave and pulsed wave (pulsatile deliveryof ultrasound) may be employed in any combination. For example,continuous wave ultrasound may be applied, followed by pulsed waveultrasound, or vice versa. This may be repeated any number of times, inany order and combination. The pulsed wave ultrasound may be appliedagainst a background of continuous wave ultrasound, and any number ofpulses may be used in any number of groups.

Preferably, the ultrasound may comprise pulsed wave ultrasound. In ahighly preferred embodiment, the ultrasound is applied at a powerdensity of 0.7 Wcm-2 or 1.25 Wcm-2 as a continuous wave. Higher powerdensities may be employed if pulsed wave ultrasound is used.

Use of ultrasound is advantageous as, like light, it may be focusedaccurately on a target. Moreover, ultrasound is advantageous as it maybe focused more deeply into tissues unlike light. It is therefore bettersuited to whole-tissue penetration (such as but not limited to a lobe ofthe liver) or whole organ (such as but not limited to the entire liveror an entire muscle, such as the heart) therapy. Another importantadvantage is that ultrasound is a non-invasive stimulus which is used ina wide variety of diagnostic and therapeutic applications. By way ofexample, ultrasound is well known in medical imaging techniques and,additionally, in orthopedic therapy. Furthermore, instruments suitablefor the application of ultrasound to a subject vertebrate are widelyavailable and their use is well known in the art.

In particular embodiments, the guide molecule is modified by a secondarystructure to increase the specificity of the CRISPR-Cas system and thesecondary structure can protect against exonuclease activity and allowfor 5′ additions to the guide sequence also referred to herein as aprotected guide molecule.

In one aspect, the invention provides for hybridizing a “protector RNA”to a sequence of the guide molecule, wherein the “protector RNA” is anRNA strand complementary to the 3′ end of the guide molecule to therebygenerate a partially double-stranded guide RNA. In an embodiment of theinvention, protecting mismatched bases (i.e. the bases of the guidemolecule which do not form part of the guide sequence) with a perfectlycomplementary protector sequence decreases the likelihood of target RNAbinding to the mismatched basepairs at the 3′ end. In particularembodiments of the invention, additional sequences comprising anextended length may also be present within the guide molecule such thatthe guide comprises a protector sequence within the guide molecule. This“protector sequence” ensures that the guide molecule comprises a“protected sequence” in addition to an “exposed sequence” (comprisingthe part of the guide sequence hybridizing to the target sequence). Inparticular embodiments, the guide molecule is modified by the presenceof the protector guide to comprise a secondary structure such as ahairpin. Advantageously there are three or four to thirty or more, e.g.,about 10 or more, contiguous base pairs having complementarity to theprotected sequence, the guide sequence or both. It is advantageous thatthe protected portion does not impede thermodynamics of the CRISPR-Cassystem interacting with its target. By providing such an extensionincluding a partially double stranded guide molecule, the guide moleculeis considered protected and results in improved specific binding of theCRISPR-Cas complex, while maintaining specific activity.

In particular embodiments, use is made of a truncated guide (tru-guide),i.e. a guide molecule which comprises a guide sequence which istruncated in length with respect to the canonical guide sequence length.As described by Nowak et al. (Nucleic Acids Res (2016) 44 (20):9555-9564), such guides may allow catalytically active CRISPR-Cas enzymeto bind its target without cleaving the target RNA. In particularembodiments, a truncated guide is used which allows the binding of thetarget but retains only nickase activity of the CRISPR-Cas enzyme.

CRISPR RNA-Targeting Effector Proteins

In one example embodiment, the CRISPR system effector protein is anRNA-targeting effector protein. In certain embodiments, the CRISPRsystem effector protein is a Type VI CRISPR system targeting RNA (e.g.,Cas13a, Cas13b, Cas13c or Cas13d). Example RNA-targeting effectorproteins include Cas13b and C2c2 (now known as Cas13a). It will beunderstood that the term “C2c2” herein is used interchangeably with“Cas13a”. “C2c2” is now referred to as “Cas13a”, and the terms are usedinterchangeably herein unless indicated otherwise. As used herein, theterm “Cas13” refers to any Type VI CRISPR system targeting RNA (e.g.,Cas13a, Cas13b, Cas13c or Cas13d). When the CRISPR protein is a C2c2protein, a tracrRNA is not required. C2c2 has been described inAbudayyeh et al. (2016) “C2c2 is a single-component programmableRNA-guided RNA-targeting CRISPR effector”; Science; DOI:10.1126/science.aaf5573; and Shmakov et al. (2015) “Discovery andFunctional Characterization of Diverse Class 2 CRISPR-Cas Systems”,Molecular Cell, DOI: dx.doi.org/10.1016/j.molcel.2015.10.008; which areincorporated herein in their entirety by reference. Cas13b has beendescribed in Smargon et al. (2017) “Cas13b Is a Type VI-BCRISPR-Associated RNA-Guided RNases Differentially Regulated byAccessory Proteins Csx27 and Csx28,” Molecular Cell. 65, 1-13;dx.doi.org/10.1016/j.molcel.2016.12.023., which is incorporated hereinin its entirety by reference.

In some embodiments, one or more elements of a nucleic acid-targetingsystem is derived from a particular organism comprising an endogenousCRISPR RNA-targeting system. In certain example embodiments, theeffector protein CRISPR RNA-targeting system comprises at least one HEPNdomain, including but not limited to the HEPN domains described herein,HEPN domains known in the art, and domains recognized to be HEPN domainsby comparison to consensus sequence motifs. Several such domains areprovided herein. In one non-limiting example, a consensus sequence canbe derived from the sequences of C2c2 or Cas13b orthologs providedherein. In certain example embodiments, the effector protein comprises asingle HEPN domain. In certain other example embodiments, the effectorprotein comprises two HEPN domains.

In one example embodiment, the effector protein comprise one or moreHEPN domains comprising a RxxxxH motif sequence. The RxxxxH motifsequence can be, without limitation, from a HEPN domain described hereinor a HEPN domain known in the art. RxxxxH motif sequences furtherinclude motif sequences created by combining portions of two or moreHEPN domains. As noted, consensus sequences can be derived from thesequences of the orthologs disclosed in U.S. Provisional PatentApplication 62/432,240 entitled “Novel CRISPR Enzymes and Systems,” U.S.Provisional Patent Application 62/471,710 entitled “Novel Type VI CRISPROrthologs and Systems” filed on Mar. 15, 2017, and U.S. ProvisionalPatent Application entitled “Novel Type VI CRISPR Orthologs andSystems,” labeled as attorney docket number 47627-05-2133 and filed onApr. 12, 2017.

In certain other example embodiments, the CRISPR system effector proteinis a C2c2 nuclease. The activity of C2c2 may depend on the presence oftwo HEPN domains. These have been shown to be RNase domains, i.e.nuclease (in particular an endonuclease) cutting RNA. C2c2 HEPN may alsotarget DNA, or potentially DNA and/or RNA. On the basis that the HEPNdomains of C2c2 are at least capable of binding to and, in theirwild-type form, cutting RNA, then it is preferred that the C2c2 effectorprotein has RNase function. Regarding C2c2 CRISPR systems, reference ismade to U.S. Provisional 62/351,662 filed on Jun. 17, 2016 and U.S.Provisional 62/376,377 filed on Aug. 17, 2016. Reference is also made toU.S. Provisional 62/351,803 filed on Jun. 17, 2016. Reference is alsomade to U.S. Provisional entitled “Novel Crispr Enzymes and Systems”filed Dec. 8, 2016 bearing Broad Institute No. 10035.PA4 and AttorneyDocket No. 47627.03.2133. Reference is further made to East-Seletsky etal. “Two distinct RNase activities of CRISPR-C2c2 enable guide-RNAprocessing and RNA detection” Nature doi:10/1038/nature19802 andAbudayyeh et al. “C2c2 is a single-component programmable RNA-guided RNAtargeting CRISPR effector” bioRxiv doi:10.1101/054742.

In certain embodiments, the C2c2 effector protein is from an organism ofa genus selected from the group consisting of: Leptotrichia, Listeria,Corynebacter, Sutterella, Legionella, Treponema, Filifactor,Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides,Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum,Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus,Nitratifractor, Mycoplasma, Campylobacter, and Lachnospira, or the C2c2effector protein is an organism selected from the group consisting of:Leptotrichia shahii, Leptotrichia. wadei, Listeria seeligeri,Clostridium aminophilum, Carnobacterium gallinarum, Paludibacterpropionicigenes, Listeria weihenstephanensis, or the C2c2 effectorprotein is a L. wadei F0279 or L. wadei F0279 (Lw2) C2C2 effectorprotein. In another embodiment, the one or more guide RNAs are designedto detect a single nucleotide polymorphism, splice variant of atranscript, or a frameshift mutation in a target RNA or DNA.

In certain example embodiments, the RNA-targeting effector protein is aType VI-B effector protein, such as Cas13b and Group 29 or Group 30proteins. In certain example embodiments, the RNA-targeting effectorprotein comprises one or more HEPN domains. In certain exampleembodiments, the RNA-targeting effector protein comprises a C-terminalHEPN domain, a N-terminal HEPN domain, or both. Regarding example TypeVI-B effector proteins that may be used in the context of thisinvention, reference is made to U.S. application Ser. No. 15/331,792entitled “Novel CRISPR Enzymes and Systems” and filed Oct. 21, 2016,International Patent Application No. PCT/US2016/058302 entitled “NovelCRISPR Enzymes and Systems”, and filed Oct. 21, 2016, and Smargon et al.“Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentiallyregulated by accessory proteins Csx27 and Csx28” Molecular Cell, 65,1-13 (2017); dx.doi.org/10.1016/j.molcel.2016.12.023, and U.S.Provisional Application No. to be assigned, entitled “Novel Cas13bOrthologues CRISPR Enzymes and System” filed Mar. 15, 2017. Inparticular embodiments, the Cas13b enzyme is derived from Bergeyellazoohelcum.

In certain example embodiments, the RNA-targeting effector protein is aCas13c effector protein as disclosed in U.S. Provisional PatentApplication No. 62/525,165 filed Jun. 26, 2017, and PCT Application No.US 2017/047193 filed Aug. 16, 2017.

In some embodiments, one or more elements of a nucleic acid-targetingsystem is derived from a particular organism comprising an endogenousCRISPR RNA-targeting system. In certain embodiments, the CRISPRRNA-targeting system is found in Eubacterium and Ruminococcus. Incertain embodiments, the effector protein comprises targeted andcollateral ssRNA cleavage activity. In certain embodiments, the effectorprotein comprises dual HEPN domains. In certain embodiments, theeffector protein lacks a counterpart to the Helical-1 domain of Cas13a.In certain embodiments, the effector protein is smaller than previouslycharacterized class 2 CRISPR effectors, with a median size of 928 aa.This median size is 190 aa (17%) less than that of Cas13c, more than 200aa (18%) less than that of Cas13b, and more than 300 aa (26%) less thanthat of Cas13a. In certain embodiments, the effector protein has norequirement for a flanking sequence (e.g., PFS, PAM).

In certain embodiments, the effector protein locus structures include aWYL domain containing accessory protein (so denoted after three aminoacids that were conserved in the originally identified group of thesedomains; see, e.g., WYL domain IPR026881). In certain embodiments, theWYL domain accessory protein comprises at least one helix-turn-helix(HTH) or ribbon-helix-helix (RHH) DNA-binding domain. In certainembodiments, the WYL domain containing accessory protein increases boththe targeted and the collateral ssRNA cleavage activity of theRNA-targeting effector protein. In certain embodiments, the WYL domaincontaining accessory protein comprises an N-terminal RHH domain, as wellas a pattern of primarily hydrophobic conserved residues, including aninvariant tyrosine-leucine doublet corresponding to the original WYLmotif. In certain embodiments, the WYL domain containing accessoryprotein is WYL1. WYL1 is a single WYL-domain protein associatedprimarily with Ruminococcus.

In other example embodiments, the Type VI RNA-targeting Cas enzyme isCas13d. In certain embodiments, Cas13d is Eubacterium siraeum DSM 15702(EsCas13d) or Ruminococcus sp. N15.MGS-57 (RspCas13d) (see, e.g., Yan etal., Cas13d Is a Compact RNA-Targeting Type VI CRISPR EffectorPositively Modulated by a WYL-Domain-Containing Accessory Protein,Molecular Cell (2018), doi.org/10.1016/j.molcel.2018.02.028). RspCas13dand EsCas13d have no flanking sequence requirements (e.g., PFS, PAM).

Cas13 RNA Editing

In one aspect, the invention provides a method of modifying or editing atarget transcript in a eukaryotic cell. In some embodiments, the methodcomprises allowing a CRISPR-Cas effector module complex to bind to thetarget polynucleotide to effect RNA base editing, wherein the CRISPR-Caseffector module complex comprises a Cas effector module complexed with aguide sequence hybridized to a target sequence within said targetpolynucleotide, wherein said guide sequence is linked to a direct repeatsequence. In some embodiments, the Cas effector module comprises acatalytically inactive CRISPR-Cas protein. In some embodiments, theguide sequence is designed to introduce one or more mismatches to theRNA/RNA duplex formed between the target sequence and the guidesequence. In particular embodiments, the mismatch is an A-C mismatch. Insome embodiments, the Cas effector may associate with one or morefunctional domains (e.g. via fusion protein or suitable linkers). Insome embodiments, the effector domain comprises one or more cytindine oradenosine deaminases that mediate endogenous editing of via hydrolyticdeamination. In particular embodiments, the effector domain comprisesthe adenosine deaminase acting on RNA (ADAR) family of enzymes. Inparticular embodiments, the adenosine deaminase protein or catalyticdomain thereof capable of deaminating adenosine or cytidine in RNA or isan RNA specific adenosine deaminase and/or is a bacterial, human,cephalopod, or Drosophila adenosine deaminase protein or catalyticdomain thereof, preferably TadA, more preferably ADAR, optionallyhuADAR, optionally (hu)ADAR1 or (hu)ADAR2, preferably huADAR2 orcatalytic domain thereof.

The present application relates to modifying a target RNA sequence ofinterest (see, e.g, Cox et al., Science. 2017 Nov. 24;358(6366):1019-1027). Using RNA-targeting rather than DNA targetingoffers several advantages relevant for therapeutic development. First,there are substantial safety benefits to targeting RNA: there will befewer off-target events because the available sequence space in thetranscriptome is significantly smaller than the genome, and if anoff-target event does occur, it will be transient and less likely toinduce negative side effects. Second, RNA-targeting therapeutics will bemore efficient because they are cell-type independent and not have toenter the nucleus, making them easier to deliver.

A further aspect of the invention relates to the method and compositionas envisaged herein for use in prophylactic or therapeutic treatment,preferably wherein said target locus of interest is within a human oranimal and to methods of modifying an Adenine or Cytidine in a targetRNA sequence of interest, comprising delivering to said target RNA, thecomposition as described herein. In particular embodiments, the CRISPRsystem and the adenonsine deaminase, or catalytic domain thereof, aredelivered as one or more polynucleotide molecules, as aribonucleoprotein complex, optionally via particles, vesicles, or one ormore viral vectors. In particular embodiments, the invention thuscomprises compositions for use in therapy. This implies that the methodscan be performed in vivo, ex vivo or in vitro. In particularembodiments, when the target is a human or animal target, the method iscarried out ex vivo or in vitro.

A further aspect of the invention relates to the method as envisagedherein for use in prophylactic or therapeutic treatment, preferablywherein said target of interest is within a human or animal and tomethods of modifying an Adenine or Cytidine in a target RNA sequence ofinterest, comprising delivering to said target RNA, the composition asdescribed herein. In particular embodiments, the CRISPR system and theadenonsine deaminase, or catalytic domain thereof, are delivered as oneor more polynucleotide molecules, as a ribonucleoprotein complex,optionally via particles, vesicles, or one or more viral vectors.

In one aspect, the invention provides a method of generating aeukaryotic cell comprising a modified or edited gene. In someembodiments, the method comprises (a) introducing one or more vectorsinto a eukaryotic cell, wherein the one or more vectors drive expressionof one or more of: Cas effector module, and a guide sequence linked to adirect repeat sequence, wherein the Cas effector module associate one ormore effector domains that mediate base editing, and (b) allowing aCRISPR-Cas effector module complex to bind to a target polynucleotide toeffect base editing of the target polynucleotide within said diseasegene, wherein the CRISPR-Cas effector module complex comprises a Caseffector module complexed with the guide sequence that is hybridized tothe target sequence within the target polynucleotide, wherein the guidesequence may be designed to introduce one or more mismatches between theRNA/RNA duplex formed between the guide sequence and the targetsequence. In particular embodiments, the mismatch is an A-C mismatch. Insome embodiments, the Cas effector may associate with one or morefunctional domains (e.g. via fusion protein or suitable linkers). Insome embodiments, the effector domain comprises one or more cytidine oradenosine deaminases that mediate endogenous editing of via hydrolyticdeamination. In particular embodiments, the effector domain comprisesthe adenosine deaminase acting on RNA (ADAR) family of enzymes. Inparticular embodiments, the adenosine deaminase protein or catalyticdomain thereof capable of deaminating adenosine or cytidine in RNA or isan RNA specific adenosine deaminase and/or is a bacterial, human,cephalopod, or Drosophila adenosine deaminase protein or catalyticdomain thereof, preferably TadA, more preferably ADAR, optionallyhuADAR, optionally (hu)ADAR1 or (hu)ADAR2, preferably huADAR2 orcatalytic domain thereof.

A further aspect relates to an isolated cell obtained or obtainable fromthe methods described herein comprising the composition described hereinor progeny of said modified cell, preferably wherein said cell comprisesa hypoxanthine or a guanine in replace of said Adenine in said targetRNA of interest compared to a corresponding cell not subjected to themethod. In particular embodiments, the cell is a eukaryotic cell,preferably a human or non-human animal cell, optionally a therapeutic Tcell or an antibody-producing B-cell.

In some embodiments, the modified cell is a therapeutic T cell, such asa T cell suitable for adoptive cell transfer therapies (e.g., CAR-Ttherapies). The modification may result in one or more desirable traitsin the therapeutic T cell, as described further herein.

The invention further relates to a method for cell therapy, comprisingadministering to a patient in need thereof the modified cell describedherein, wherein the presence of the modified cell remedies a disease inthe patient. In one embodiment, the modified cell for cell therapy is aCAR-T cell capable of recognizing and/or attacking a tumor cell.

Cytosine Deaminase

Programmable deamination of cytosine has been reported and may be usedfor correction of A→G and T→C point mutations. For example, Komor etal., Nature (2016) 533:420-424 reports targeted deamination of cytosineby APOBEC1 cytidine deaminase in a non-targeted DNA stranded displacedby the binding of a Cas9-guide RNA complex to a targeted DNA strand,which results in conversion of cytosine to uracil. See also Kim et al.,Nature Biotechnology (2017) 35:371-376; Shimatani et al., NatureBiotechnology (2017) doi:10.1038/nbt.3833; Zong et al., NatureBiotechnology (2017) doi:10.1038/nbt.3811; Yang Nature Communication(2016) doi:10.1038/ncomms13330.

Adenosine Deaminase

The term “adenosine deaminase” or “adenosine deaminase protein” as usedherein refers to a protein, a polypeptide, or one or more functionaldomain(s) of a protein or a polypeptide that is capable of catalyzing ahydrolytic deamination reaction that converts an adenine (or an adeninemoiety of a molecule) to a hypoxanthine (or a hypoxanthine moiety of amolecule), as shown below. In some embodiments, the adenine-containingmolecule is an adenosine (A), and the hypoxanthine-containing moleculeis an inosine (I). The adenine-containing molecule can bedeoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

According to the present disclosure, adenosine deaminases that can beused in connection with the present disclosure include, but are notlimited to, members of the enzyme family known as adenosine deaminasesthat act on RNA (ADARs), members of the enzyme family known as adenosinedeaminases that act on tRNA (ADATs), and other adenosine deaminasedomain-containing (ADAD) family members. According to the presentdisclosure, the adenosine deaminase is capable of targeting adenine in aRNA/DNA heteroduplex. Indeed, Zheng et al. (Nucleic Acids Res. 2017,45(6): 3369-3377) has demonstrated that ADARs can carry out adenosine toinosine editing reactions on RNA/DNA heteroduplexes. In particularembodiments, the adenosine deaminase has been modified to increase itsability to edit DNA in a RNA/DNA heteroduplex as detailed herein.

In some embodiments, the adenosine deaminase is derived from one or moremetazoa species, including but not limited to, mammals, birds, frogs,squids, fish, flies and worms. In some embodiments, the adenosinedeaminase is a human, squid or Drosophila adenosine deaminase.

In some embodiments, the adenosine deaminase is a human ADAR, includinghADAR1, hADAR2, hADAR3. In some embodiments, the adenosine deaminase isa Caenorhabditis elegans ADAR protein, including ADR-1 and ADR-2. Insome embodiments, the adenosine deaminase is a Drosophila ADAR protein,including dAdar. In some embodiments, the adenosine deaminase is a squidLoligo pealeii ADAR protein, including sqADAR2a and sqADAR2b. In someembodiments, the adenosine deaminase is a human ADAT protein. In someembodiments, the adenosine deaminase is a Drosophila ADAT protein. Insome embodiments, the adenosine deaminase is a human ADAD protein,including TENR (hADAD1) and TENRL (hADAD2).

In some embodiments, the adenosine deaminase protein recognizes andconverts one or more target adenosine residue(s) in a double-strandednucleic acid substrate into inosine residues (s). In some embodiments,the double-stranded nucleic acid substrate is a RNA-DNA hybrid duplex.In some embodiments, the adenosine deaminase protein recognizes abinding window on the double-stranded substrate. In some embodiments,the binding window contains at least one target adenosine residue(s). Insome embodiments, the binding window is in the range of about 3 bp toabout 100 bp. In some embodiments, the binding window is in the range ofabout 5 bp to about 50 bp. In some embodiments, the binding window is inthe range of about 10 bp to about 30 bp. In some embodiments, thebinding window is about 1 bp, 2 bp, 3 bp, 5 bp, 7 bp, 10 bp, 15 bp, 20bp, 25 bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75bp, 80 bp, 85 bp, 90 bp, 95 bp, or 100 bp.

In some embodiments, the adenosine deaminase protein comprises one ormore deaminase domains. Not intended to be bound by theory, it iscontemplated that the deaminase domain functions to recognize andconvert one or more target adenosine (A) residue(s) contained in adouble-stranded nucleic acid substrate into inosine (I) residues (s). Insome embodiments, the deaminase domain comprises an active center. Insome embodiments, the active center comprises a zinc ion. In someembodiments, during the A-to-I editing process, base pairing at thetarget adenosine residue is disrupted, and the target adenosine residueis “flipped” out of the double helix to become accessible by theadenosine deaminase. In some embodiments, amino acid residues in or nearthe active center interact with one or more nucleotide(s) 5′ to a targetadenosine residue. In some embodiments, amino acid residues in or nearthe active center interact with one or more nucleotide(s) 3′ to a targetadenosine residue. In some embodiments, amino acid residues in or nearthe active center further interact with the nucleotide complementary tothe target adenosine residue on the opposite strand. In someembodiments, the amino acid residues form hydrogen bonds with the 2′hydroxyl group of the nucleotides.

In some embodiments, the adenosine deaminase comprises human ADAR2 fullprotein (hADAR2) or the deaminase domain thereof (hADAR2-D). In someembodiments, the adenosine deaminase is an ADAR family member that ishomologous to hADAR2 or hADAR2-D.

Particularly, in some embodiments, the homologous ADAR protein is humanADAR1 (hADAR1) or the deaminase domain thereof (hADAR1-D). In someembodiments, glycine 1007 of hADAR1-D corresponds to glycine⁴⁸⁷hADAR2-D, and glutamic Acid¹⁰⁰⁸ of hADAR1-D corresponds to glutamicacid⁴⁸⁸ of hADAR2-D.

In some embodiments, the adenosine deaminase comprises the wild-typeamino acid sequence of hADAR2-D. In some embodiments, the adenosinedeaminase comprises one or more mutations in the hADAR2-D sequence, suchthat the editing efficiency, and/or substrate editing preference ofhADAR2-D is changed according to specific needs.

Certain mutations of hADAR1 and hADAR2 proteins have been described inKuttan et al., Proc Natl Acad Sci USA. (2012) 109(48):E3295-304; Want etal. ACS Chem Biol. (2015) 10(11):2512-9; and Zheng et al. Nucleic AcidsRes. (2017) 45(6):3369-337, each of which is incorporated herein byreference in its entirety.

In some embodiments, the adenosine deaminase comprises a mutation atglycine³³⁶ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glycineresidue at position 336 is replaced by an aspartic acid residue (G336D).

In some embodiments, the adenosine deaminase comprises a mutation atGlycine⁴⁸⁷ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glycineresidue at position 487 is replaced by a non-polar amino acid residuewith relatively small side chains. For example, in some embodiments, theglycine residue at position 487 is replaced by an alanine residue(G487A). In some embodiments, the glycine residue at position 487 isreplaced by a valine residue (G487V). In some embodiments, the glycineresidue at position 487 is replaced by an amino acid residue withrelatively large side chains. In some embodiments, the glycine residueat position 487 is replaced by a arginine residue (G487R). In someembodiments, the glycine residue at position 487 is replaced by a lysineresidue (G487K). In some embodiments, the glycine residue at position487 is replaced by a tryptophan residue (G487W). In some embodiments,the glycine residue at position 487 is replaced by a tyrosine residue(G487Y).

In some embodiments, the adenosine deaminase comprises a mutation atglutamic acid⁴⁸⁸ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glutamicacid residue at position 488 is replaced by a glutamine residue (E488Q).In some embodiments, the glutamic acid residue at position 488 isreplaced by a histidine residue (E488H). In some embodiments, theglutamic acid residue at position 488 is replace by an arginine residue(E488R). In some embodiments, the glutamic acid residue at position 488is replace by a lysine residue (E488K). In some embodiments, theglutamic acid residue at position 488 is replace by an asparagineresidue (E488N). In some embodiments, the glutamic acid residue atposition 488 is replace by an alanine residue (E488A). In someembodiments, the glutamic acid residue at position 488 is replace by aMethionine residue (E488M). In some embodiments, the glutamic acidresidue at position 488 is replace by a serine residue (E488S). In someembodiments, the glutamic acid residue at position 488 is replace by aphenylalanine residue (E488F). In some embodiments, the glutamic acidresidue at position 488 is replace by a lysine residue (E488L). In someembodiments, the glutamic acid residue at position 488 is replace by atryptophan residue (E488W).

In some embodiments, the adenosine deaminase comprises a mutation atthreonine⁴⁹⁰ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, thethreonine residue at position 490 is replaced by a cysteine residue(T490C). In some embodiments, the threonine residue at position 490 isreplaced by a serine residue (T490S). In some embodiments, the threonineresidue at position 490 is replaced by an alanine residue (T490A). Insome embodiments, the threonine residue at position 490 is replaced by aphenylalanine residue (T490F). In some embodiments, the threonineresidue at position 490 is replaced by a tyrosine residue (T490Y). Insome embodiments, the threonine residue at position 490 is replaced by aserine residue (T490R). In some embodiments, the threonine residue atposition 490 is replaced by an alanine residue (T490K). In someembodiments, the threonine residue at position 490 is replaced by aphenylalanine residue (T490P). In some embodiments, the threonineresidue at position 490 is replaced by a tyrosine residue (T490E).

In some embodiments, the adenosine deaminase comprises a mutation atvaline⁴⁹³ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the valineresidue at position 493 is replaced by an alanine residue (V493A). Insome embodiments, the valine residue at position 493 is replaced by aserine residue (V493S). In some embodiments, the valine residue atposition 493 is replaced by a threonine residue (V493T). In someembodiments, the valine residue at position 493 is replaced by anarginine residue (V493R). In some embodiments, the valine residue atposition 493 is replaced by an aspartic acid residue (V493D). In someembodiments, the valine residue at position 493 is replaced by a prolineresidue (V493P). In some embodiments, the valine residue at position 493is replaced by a glycine residue (V493G).

In some embodiments, the adenosine deaminase comprises a mutation atalanine⁵⁸⁹ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the alanineresidue at position 589 is replaced by a valine residue (A589V).

In some embodiments, the adenosine deaminase comprises a mutation atasparagine⁵⁹⁷ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, theasparagine residue at position 597 is replaced by a lysine residue(N597K). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by an arginine residue(N597R). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by an alanine residue(N597A). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by a glutamic acidresidue (N597E). In some embodiments, the adenosine deaminase comprisesa mutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by a histidine residue(N597H). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by a glycine residue(N597G). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by a tyrosine residue(N597Y). In some embodiments, the asparagine residue at position 597 isreplaced by a phenylalanine residue (N597F).

In some embodiments, the adenosine deaminase comprises a mutation atserine⁵⁹⁹ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the serineresidue at position 599 is replaced by a threonine residue (S599T).

In some embodiments, the adenosine deaminase comprises a mutation atasparagine⁶¹³ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, theasparagine residue at position 613 is replaced by a lysine residue(N613K). In some embodiments, the adenosine deaminase comprises amutation at position 613 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 613 is replaced by an arginine residue(N613R). In some embodiments, the adenosine deaminase comprises amutation at position 613 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 613 is replaced by an alanine residue(N613A) In some embodiments, the adenosine deaminase comprises amutation at position 613 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 613 is replaced by a glutamic acidresidue (N613E).

In some embodiments, to improve editing efficiency, the adenosinedeaminase may comprise one or more of the mutations: G336D, G487A,G487V, E488Q, E488H, E488R, E488N, E488A, E488S, E488M, T490C, T490S,V493T, V493S, V493A, V493R, V493D, V493P, V493G, N597K, N597R, N597A,N597E, N597H, N597G, N597Y, A589V, S599T, N613K, N613R, N613A, N613E,based on amino acid sequence positions of hADAR2-D, and mutations in ahomologous ADAR protein corresponding to the above.

In some embodiments, to reduce editing efficiency, the adenosinedeaminase may comprise one or more of the mutations: E488F, E488L,E488W, T490A, T490F, T490Y, T490R, T490K, T490P, T490E, N597F, based onamino acid sequence positions of hADAR2-D, and mutations in a homologousADAR protein corresponding to the above. In particular embodiments, itcan be of interest to use an adenosine deaminase enzyme with reducedefficacy to reduce off-target effects.

The terms “editing specificity” and “editing preference” are usedinterchangeably herein to refer to the extent of A-to-I editing at aparticular adenosine site in a double-stranded substrate. In someembodiment, the substrate editing preference is determined by the 5′nearest neighbor and/or the 3′ nearest neighbor of the target adenosineresidue. In some embodiments, the adenosine deaminase has preference forthe 5′ nearest neighbor of the substrate ranked as U>A>C>G (“>”indicates greater preference). In some embodiments, the adenosinedeaminase has preference for the 3′ nearest neighbor of the substrateranked as G>C-A>U (“>” indicates greater preference; “˜” indicatessimilar preference). In some embodiments, the adenosine deaminase haspreference for the 3′ nearest neighbor of the substrate ranked asG>C>U-A (“>” indicates greater preference; “˜” indicates similarpreference). In some embodiments, the adenosine deaminase has preferencefor the 3′ nearest neighbor of the substrate ranked as G>C>A>U (“>”indicates greater preference). In some embodiments, the adenosinedeaminase has preference for the 3′ nearest neighbor of the substrateranked as C-G-A>U (“>” indicates greater preference; “˜” indicatessimilar preference). In some embodiments, the adenosine deaminase haspreference for a triplet sequence containing the target adenosineresidue ranked as TAG>AAG>CAC>AAT>GAA>GAC (“>” indicates greaterpreference), the center A being the target adenosine residue.

In some embodiments, the substrate editing preference of an adenosinedeaminase is affected by the presence or absence of a nucleic acidbinding domain in the adenosine deaminase protein. In some embodiments,to modify substrate editing preference, the deaminase domain isconnected with a double-strand RNA binding domain (dsRBD) or adouble-strand RNA binding motif (dsRBM). In some embodiments, the dsRBDor dsRBM may be derived from an ADAR protein, such as hADAR1 or hADAR2.In some embodiments, a full length ADAR protein that comprises at leastone dsRBD and a deaminase domain is used. In some embodiments, the oneor more dsRBM or dsRBD is at the N-terminus of the deaminase domain. Inother embodiments, the one or more dsRBM or dsRBD is at the C-terminusof the deaminase domain.

In some embodiments, the substrate editing preference of an adenosinedeaminase is affected by amino acid residues near or in the activecenter of the enzyme. In some embodiments, to modify substrate editingpreference, the adenosine deaminase may comprise one or more of themutations: G336D, G487R, G487K, G487W, G487Y, E488Q, E488N, T490A,V493A, V493T, V493S, N597K, N597R, A589V, S599T, N613K, N613R, based onamino acid sequence positions of hADAR2-D, and mutations in a homologousADAR protein corresponding to the above.

Particularly, in some embodiments, to reduce editing specificity, theadenosine deaminase can comprise one or more of mutations E488Q, V493A,N597K, N613K, based on amino acid sequence positions of hADAR2-D, andmutations in a homologous ADAR protein corresponding to the above. Insome embodiments, to increase editing specificity, the adenosinedeaminase can comprise mutation T490A.

In some embodiments, to increase editing preference for target adenosine(A) with an immediate 5′ G, such as substrates comprising the tripletsequence GAC, the center A being the target adenosine residue, theadenosine deaminase can comprise one or more of mutations G336D, E488Q,E488N, V493T, V493S, V493A, A589V, N597K, N597R, S599T, N613K, N613R,based on amino acid sequence positions of hADAR2-D, and mutations in ahomologous ADAR protein corresponding to the above.

Particularly, in some embodiments, the adenosine deaminase comprisesmutation E488Q or a corresponding mutation in a homologous ADAR proteinfor editing substrates comprising the following triplet sequences: GAC,GAA, GAU, GAG, CAU, AAU, UAC, the center A being the target adenosineresidue.

In some embodiments, to reduce off-target effects, the adenosinedeaminase comprises one or more of mutations at R348, V351, T375, K376,E396, C451, R455, N473, R474, K475, R477, R481, 5486, E488, T490, 5495,R510, based on amino acid sequence positions of hADAR2-D, and mutationsin a homologous ADAR protein corresponding to the above. In someembodiments, the adenosine deaminase comprises mutation at E488 and oneor more additional positions selected from R348, V351, T375, K376, E396,C451, R455, N473, R474, K475, R477, R481, S486, T490, S495, R510. Insome embodiments, the adenosine deaminase comprises mutation at T375,and optionally at one or more additional positions. In some embodiments,the adenosine deaminase comprises mutation at N473, and optionally atone or more additional positions. In some embodiments, the adenosinedeaminase comprises mutation at V351, and optionally at one or moreadditional positions. In some embodiments, the adenosine deaminasecomprises mutation at E488 and T375, and optionally at one or moreadditional positions. In some embodiments, the adenosine deaminasecomprises mutation at E488 and N473, and optionally at one or moreadditional positions. In some embodiments, the adenosine deaminasecomprises mutation E488 and V351, and optionally at one or moreadditional positions. In some embodiments, the adenosine deaminasecomprises mutation at E488 and one or more of T375, N473, and V351.

In some embodiments, to reduce off-target effects, the adenosinedeaminase comprises one or more of mutations selected from R348E, V351L,T375G, T375S, R455G, R455S, R455E, N473D, R474E, K475Q, R477E, R481E,S486T, E488Q, T490A, T490S, S495T, and R510E, based on amino acidsequence positions of hADAR2-D, and mutations in a homologous ADARprotein corresponding to the above. In some embodiments, the adenosinedeaminase comprises mutation E488Q and one or more additional mutationsselected from R348E, V351L, T375G, T375S, R455G, R455S, R455E, N473D,R474E, K475Q, R477E, R481E, S486T, T490A, T490S, S495T, and R510E. Insome embodiments, the adenosine deaminase comprises mutation T375G orT375S, and optionally one or more additional mutations. In someembodiments, the adenosine deaminase comprises mutation N473D, andoptionally one or more additional mutations. In some embodiments, theadenosine deaminase comprises mutation V351L, and optionally one or moreadditional mutations. In some embodiments, the adenosine deaminasecomprises mutation E488Q, and T375G or T375G, and optionally one or moreadditional mutations. In some embodiments, the adenosine deaminasecomprises mutation E488Q and N473D, and optionally one or moreadditional mutations. In some embodiments, the adenosine deaminasecomprises mutation E488Q and V351L, and optionally one or moreadditional mutations. In some embodiments, the adenosine deaminasecomprises mutation E488Q and one or more of T375G/S, N473D and V351L.

Crystal structures of the human ADAR2 deaminase domain bound to duplexRNA reveal a protein loop that binds the RNA on the 5′ side of themodification site. This 5′ binding loop is one contributor to substratespecificity differences between ADAR family members. See Wang et al.,Nucleic Acids Res., 44(20):9872-9880 (2016), the content of which isincorporated herein by reference in its entirety. In addition, anADAR2-specific RNA-binding loop was identified near the enzyme activesite. See Mathews et al., Nat. Struct. Mol. Biol., 23(5):426-33 (2016),the content of which is incorporated herein by reference in itsentirety. In some embodiments, the adenosine deaminase comprises one ormore mutations in the RNA binding loop to improve editing specificityand/or efficiency.

In some embodiments, the adenosine deaminase comprises a mutation atalanine⁴⁵⁴ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the alanineresidue at position 454 is replaced by a serine residue (A4545). In someembodiments, the alanine residue at position 454 is replaced by acysteine residue (A454C). In some embodiments, the alanine residue atposition 454 is replaced by an aspartic acid residue (A454D).

In some embodiments, the adenosine deaminase comprises a mutation atarginine⁴⁵⁵ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the arginineresidue at position 455 is replaced by an alanine residue (R455A). Insome embodiments, the arginine residue at position 455 is replaced by avaline residue (R455V). In some embodiments, the arginine residue atposition 455 is replaced by a histidine residue (R455H). In someembodiments, the arginine residue at position 455 is replaced by aglycine residue (R455G). In some embodiments, the arginine residue atposition 455 is replaced by a serine residue (R455S). In someembodiments, the arginine residue at position 455 is replaced by aglutamic acid residue (R455E).

In some embodiments, the adenosine deaminase comprises a mutation atisoleucine⁴⁵⁶ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, theisoleucine residue at position 456 is replaced by a valine residue(I456V). In some embodiments, the isoleucine residue at position 456 isreplaced by a leucine residue (I456L). In some embodiments, theisoleucine residue at position 456 is replaced by an aspartic acidresidue (I456D).

In some embodiments, the adenosine deaminase comprises a mutation atphenylalanine⁴⁵⁷ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, thephenylalanine residue at position 457 is replaced by a tyrosine residue(F457Y). In some embodiments, the phenylalanine residue at position 457is replaced by an arginine residue (F457R). In some embodiments, thephenylalanine residue at position 457 is replaced by a glutamic acidresidue (F457E).

In some embodiments, the adenosine deaminase comprises a mutation atserine⁴⁵⁸ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the serineresidue at position 458 is replaced by a valine residue (S458V). In someembodiments, the serine residue at position 458 is replaced by aphenylalanine residue (S458F). In some embodiments, the serine residueat position 458 is replaced by a proline residue (S458P).

In some embodiments, the adenosine deaminase comprises a mutation atproline⁴⁵⁹ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the prolineresidue at position 459 is replaced by a cysteine residue (P459C). Insome embodiments, the proline residue at position 459 is replaced by ahistidine residue (P459H). In some embodiments, the proline residue atposition 459 is replaced by a tryptophan residue (P459W).

In some embodiments, the adenosine deaminase comprises a mutation athistidine⁴⁶⁰ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, thehistidine residue at position 460 is replaced by an arginine residue(H460R). In some embodiments, the histidine residue at position 460 isreplaced by an isoleucine residue (H460I). In some embodiments, thehistidine residue at position 460 is replaced by a proline residue(H460P).

In some embodiments, the adenosine deaminase comprises a mutation atproline⁴⁶² of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the prolineresidue at position 462 is replaced by a serine residue (P462S). In someembodiments, the proline residue at position 462 is replaced by atryptophan residue (P462W). In some embodiments, the proline residue atposition 462 is replaced by a glutamic acid residue (P462E).

In some embodiments, the adenosine deaminase comprises a mutation ataspartic acid⁴⁶⁹ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the asparticacid residue at position 469 is replaced by a glutamine residue (D469Q).In some embodiments, the aspartic acid residue at position 469 isreplaced by a serine residue (D469S). In some embodiments, the asparticacid residue at position 469 is replaced by a tyrosine residue (D469Y).

In some embodiments, the adenosine deaminase comprises a mutation atarginine⁴⁷⁰ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the arginineresidue at position 470 is replaced by an alanine residue (R470A). Insome embodiments, the arginine residue at position 470 is replaced by anisoleucine residue (R470I). In some embodiments, the arginine residue atposition 470 is replaced by an aspartic acid residue (R470D).

In some embodiments, the adenosine deaminase comprises a mutation athistidine⁴⁷¹ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, thehistidine residue at position 471 is replaced by a lysine residue(H471K). In some embodiments, the histidine residue at position 471 isreplaced by a threonine residue (H471T). In some embodiments, thehistidine residue at position 471 is replaced by a valine residue(H471V).

In some embodiments, the adenosine deaminase comprises a mutation atproline⁴⁷² of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the prolineresidue at position 472 is replaced by a lysine residue (P472K). In someembodiments, the proline residue at position 472 is replaced by athreonine residue (P472T). In some embodiments, the proline residue atposition 472 is replaced by an aspartic acid residue (P472D).

In some embodiments, the adenosine deaminase comprises a mutation atasparagine⁴⁷³ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, theasparagine residue at position 473 is replaced by an arginine residue(N473R). In some embodiments, the asparagine residue at position 473 isreplaced by a tryptophan residue (N473W). In some embodiments, theasparagine residue at position 473 is replaced by a proline residue(N473P). In some embodiments, the asparagine residue at position 473 isreplaced by an aspartic acid residue (N473D).

In some embodiments, the adenosine deaminase comprises a mutation atarginine⁴⁷⁴ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the arginineresidue at position 474 is replaced by a lysine residue (R474K). In someembodiments, the arginine residue at position 474 is replaced by aglycine residue (R474G). In some embodiments, the arginine residue atposition 474 is replaced by an aspartic acid residue (R474D). In someembodiments, the arginine residue at position 474 is replaced by aglutamic acid residue (R474E).

In some embodiments, the adenosine deaminase comprises a mutation atlysine⁴⁷⁵ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the lysineresidue at position 475 is replaced by a glutamine residue (K475Q). Insome embodiments, the lysine residue at position 475 is replaced by anasparagine residue (K475N). In some embodiments, the lysine residue atposition 475 is replaced by an aspartic acid residue (K475D).

In some embodiments, the adenosine deaminase comprises a mutation atalanine⁴⁷⁶ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the alanineresidue at position 476 is replaced by a serine residue (A476S). In someembodiments, the alanine residue at position 476 is replaced by anarginine residue (A476R). In some embodiments, the alanine residue atposition 476 is replaced by a glutamic acid residue (A476E).

In some embodiments, the adenosine deaminase comprises a mutation atarginine⁴⁷⁷ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the arginineresidue at position 477 is replaced by a lysine residue (R477K). In someembodiments, the arginine residue at position 477 is replaced by athreonine residue (R477T). In some embodiments, the arginine residue atposition 477 is replaced by a phenylalanine residue (R477F). In someembodiments, the arginine residue at position 474 is replaced by aglutamic acid residue (R477E).

In some embodiments, the adenosine deaminase comprises a mutation atglycine⁴⁷⁸ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glycineresidue at position 478 is replaced by an alanine residue (G478A). Insome embodiments, the glycine residue at position 478 is replaced by anarginine residue (G478R). In some embodiments, the glycine residue atposition 478 is replaced by a tyrosine residue (G478Y).

In some embodiments, the adenosine deaminase comprises a mutation atglutamine⁴⁷⁹ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, theglutamine residue at position 479 is replaced by an asparagine residue(Q479N). In some embodiments, the glutamine residue at position 479 isreplaced by a serine residue (Q479S). In some embodiments, the glutamineresidue at position 479 is replaced by a proline residue (Q479P).

In some embodiments, the adenosine deaminase comprises a mutation atarginine³⁴⁸ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the arginineresidue at position 348 is replaced by an alanine residue (R348A). Insome embodiments, the arginine residue at position 348 is replaced by aglutamic acid residue (R348E).

In some embodiments, the adenosine deaminase comprises a mutation atvaline³⁵¹ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the valineresidue at position 351 is replaced by a leucine residue (V351L).

In some embodiments, the adenosine deaminase comprises a mutation atthreonine³⁷⁵ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, thethreonine residue at position 375 is replaced by a glycine residue(T375G). In some embodiments, the threonine residue at position 375 isreplaced by a serine residue (T375S).

In some embodiments, the adenosine deaminase comprises a mutation atarginine⁴⁸¹ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the arginineresidue at position 481 is replaced by a glutamic acid residue (R481E).

In some embodiments, the adenosine deaminase comprises a mutation atserine⁴⁸⁶ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the serineresidue at position 486 is replaced by a threonine residue (S486T).

In some embodiments, the adenosine deaminase comprises a mutation atthreonine⁴⁹⁰ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, thethreonine residue at position 490 is replaced by an alanine residue(T490A). In some embodiments, the threonine residue at position 490 isreplaced by a serine residue (T490S).

In some embodiments, the adenosine deaminase comprises a mutation atserine⁴⁹⁵ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the serineresidue at position 495 is replaced by a threonine residue (S495T).

In some embodiments, the adenosine deaminase comprises a mutation atarginine⁵¹⁰ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the arginineresidue at position 510 is replaced by a glutamine residue (R510Q). Insome embodiments, the arginine residue at position 510 is replaced by analanine residue (R510A). In some embodiments, the arginine residue atposition 510 is replaced by a glutamic acid residue (R510E).

In some embodiments, the adenosine deaminase comprises a mutation atglycine⁵⁹³ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glycineresidue at position 593 is replaced by an alanine residue (G593A). Insome embodiments, the glycine residue at position 593 is replaced by aglutamic acid residue (G593E).

In some embodiments, the adenosine deaminase comprises a mutation atlysine⁵⁹⁴ of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the lysineresidue at position 594 is replaced by an alanine residue (K594A).

In some embodiments, the adenosine deaminase comprises the wild-typeamino acid sequence of hADAR1-D (e.g.,MGSGGGGSEGAPKKKRKVGSSLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKYNSQTAKDSIFEPAKGGEKLQIKKTVSFHLYISTAPCGDGALFDKSCSDRAMESTESRHYPVFENPKQGKLRTKVENGQGTIPVESSDIVPTWDGIRLGERLRTMSCSDKILRWNVLGLQGALLTHFLQPIYLKSVTLGYLFSQGHLTRAICCRVTRDGSAFEDGLRHPFIVNHPKVGRVSIYDSKRQSGKTKETSVNWCLADGYDLEILDGTRGTVDGPRNELSRVSKKNIFLLFKKLCSFRYRRDLLRLSYGEAKKAARDYETAKNYFKKGLKDMGYGNWISKPQEEKNF* (SEQ ID NO:3)). In some embodiments, theadenosine deaminase comprises one or more mutations in the hADAR1-Dsequence, such that the editing efficiency, and/or substrate editingpreference of hADAR1-D is changed according to specific needs.

In some embodiments, the adenosine deaminase comprises a mutation atGlycine¹⁰⁰⁷ of the hADAR1-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glycineresidue at position 1007 is replaced by a non-polar amino acid residuewith relatively small side chains. For example, in some embodiments, theglycine residue at position 1007 is replaced by an alanine residue(G1007A). In some embodiments, the glycine residue at position 1007 isreplaced by a valine residue (G1007V). In some embodiments, the glycineresidue at position 1007 is replaced by an amino acid residue withrelatively large side chains. In some embodiments, the glycine residueat position 1007 is replaced by an arginine residue (G1007R). In someembodiments, the glycine residue at position 1007 is replaced by alysine residue (G1007K). In some embodiments, the glycine residue atposition 1007 is replaced by a tryptophan residue (G1007W). In someembodiments, the glycine residue at position 1007 is replaced by atyrosine residue (G1007Y). Additionally, in other embodiments, theglycine residue at position 1007 is replaced by a leucine residue(G1007L). In other embodiments, the glycine residue at position 1007 isreplaced by a threonine residue (G1007T). In other embodiments, theglycine residue at position 1007 is replaced by a serine residue(G1007S).

In some embodiments, the adenosine deaminase comprises a mutation atglutamic acid¹⁰⁰⁸ of the hADAR1-D amino acid sequence, or acorresponding position in a homologous ADAR protein. In someembodiments, the glutamic acid residue at position 1008 is replaced by apolar amino acid residue having a relatively large side chain. In someembodiments, the glutamic acid residue at position 1008 is replaced by aglutamine residue (E1008Q). In some embodiments, the glutamic acidresidue at position 1008 is replaced by a histidine residue (E1008H). Insome embodiments, the glutamic acid residue at position 1008 is replacedby an arginine residue (E1008R). In some embodiments, the glutamic acidresidue at position 1008 is replaced by a lysine residue (E1008K). Insome embodiments, the glutamic acid residue at position 1008 is replacedby a nonpolar or small polar amino acid residue. In some embodiments,the glutamic acid residue at position 1008 is replaced by aphenylalanine residue (E1008F). In some embodiments, the glutamic acidresidue at position 1008 is replaced by a tryptophan residue (E1008W).In some embodiments, the glutamic acid residue at position 1008 isreplaced by a glycine residue (E1008G). In some embodiments, theglutamic acid residue at position 1008 is replaced by an isoleucineresidue (E1008I). In some embodiments, the glutamic acid residue atposition 1008 is replaced by a valine residue (E1008V). In someembodiments, the glutamic acid residue at position 1008 is replaced by aproline residue (E1008P). In some embodiments, the glutamic acid residueat position 1008 is replaced by a serine residue (E1008S). In otherembodiments, the glutamic acid residue at position 1008 is replaced byan asparagine residue (E1008N). In other embodiments, the glutamic acidresidue at position 1008 is replaced by an alanine residue (E1008A). Inother embodiments, the glutamic acid residue at position 1008 isreplaced by a Methionine residue (E1008M). In some embodiments, theglutamic acid residue at position 1008 is replaced by a leucine residue(E1008L).

In some embodiments, to improve editing efficiency, the adenosinedeaminase may comprise one or more of the mutations: E1007S, E1007A,E1007V, E1008Q, E1008R, E1008H, E1008M, E1008N, E1008K, based on aminoacid sequence positions of hADAR1-D, and mutations in a homologous ADARprotein corresponding to the above.

In some embodiments, to reduce editing efficiency, the adenosinedeaminase may comprise one or more of the mutations: E1007R, E1007K,E1007Y, E1007L, E1007T, E1008G, E1008I, E1008P, E1008V, E1008F, E1008W,E1008S, E1008N, E1008K, based on amino acid sequence positions ofhADAR1-D, and mutations in a homologous ADAR protein corresponding tothe above.

In some embodiments, the substrate editing preference, efficiency and/orselectivity of an adenosine deaminase is affected by amino acid residuesnear or in the active center of the enzyme. In some embodiments, theadenosine deaminase comprises a mutation at the glutamic acid 1008position in hADAR1-D sequence, or a corresponding position in ahomologous ADAR protein. In some embodiments, the mutation is E1008R, ora corresponding mutation in a homologous ADAR protein. In someembodiments, the E1008R mutant has an increased editing efficiency fortarget adenosine residue that has a mismatched G residue on the oppositestrand.

In some embodiments, the adenosine deaminase protein further comprisesor is connected to one or more double-stranded RNA (dsRNA) bindingmotifs (dsRBMs) or domains (dsRBDs) for recognizing and binding todouble-stranded nucleic acid substrates. In some embodiments, theinteraction between the adenosine deaminase and the double-strandedsubstrate is mediated by one or more additional protein factor(s),including a CRISPR/CAS protein factor. In some embodiments, theinteraction between the adenosine deaminase and the double-strandedsubstrate is further mediated by one or more nucleic acid component(s),including a guide RNA.

According to the present invention, the substrate of the adenosinedeaminase is an RNA/DNA heteroduplex formed upon binding of the guidemolecule to its DNA target which then forms the CRISPR-Cas complex withthe CRISPR-Cas enzyme. The RNA/DNA or DNA/RNA heteroduplex is alsoreferred to herein as the “RNA/DNA hybrid”, “DNA/RNA hybrid” or“double-stranded substrate”. The particular features of the guidemolecule and CRISPR-Cas enzyme are detailed below.

The term “editing selectivity” as used herein refers to the fraction ofall sites on a double-stranded substrate that is edited by an adenosinedeaminase. Without being bound by theory, it is contemplated thatediting selectivity of an adenosine deaminase is affected by thedouble-stranded substrate's length and secondary structures, such as thepresence of mismatched bases, bulges and/or internal loops.

In some embodiments, when the substrate is a perfectly base-pairedduplex longer than 50 bp, the adenosine deaminase may be able todeaminate multiple adenosine residues within the duplex (e.g., 50% ofall adenosine residues). In some embodiments, when the substrate isshorter than 50 bp, the editing selectivity of an adenosine deaminase isaffected by the presence of a mismatch at the target adenosine site.Particularly, in some embodiments, adenosine (A) residue having amismatched cytidine (C) residue on the opposite strand is deaminatedwith high efficiency. In some embodiments, adenosine (A) residue havinga mismatched guanosine (G) residue on the opposite strand is skippedwithout editing.

The present invention may be further illustrated and extended based onaspects of CRISPR-Cas development and use as set forth in the followingarticles and particularly as relates to delivery of a CRISPR proteincomplex and uses of an RNA guided endonuclease in cells and organisms:

-   -   Multiplex genome engineering using CRISPR-Cas systems. Cong, L.,        Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.        D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science        February 15; 339(6121):819-23 (2013);    -   RNA-guided editing of bacterial genomes using CRISPR-Cas        systems. Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A.        Nat Biotechnol March; 31(3):233-9 (2013);    -   One-Step Generation of Mice Carrying Mutations in Multiple Genes        by CRISPR-Cas-Mediated Genome Engineering. Wang H., Yang H.,        Shivalila CS., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R.        Cell May 9; 153(4):910-8 (2013);    -   Optical control of mammalian endogenous transcription and        epigenetic states. Konermann S, Brigham M D, Trevino A E, Hsu P        D, Heidenreich M, Cong L, Platt R J, Scott D A, Church G M,        Zhang F. Nature. August 22; 500(7463):472-6. doi:        10.1038/Nature12466. Epub 2013 Aug. 23 (2013);    -   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome        Editing Specificity. Ran, F A., Hsu, P D., Lin, C Y.,        Gootenberg, J S., Konermann, S., Trevino, A E., Scott, D A.,        Inoue, A., Matoba, S., Zhang, Y., & Zhang, F. Cell August 28.        pii: S0092-8674(13)01015-5 (2013-A);    -   DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,        Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala,        V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J.,        Marraffini, L A., Bao, G., & Zhang, F. Nat Biotechnol        doi:10.1038/nbt.2647 (2013);    -   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu,        P D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature        Protocols November; 8(11):2281-308 (2013-B);    -   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.        Shalem, O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A.,        Mikkelson, T., Heckl, D., Ebert, B L., Root, D E., Doench, J G.,        Zhang, F. Science December 12. (2013);    -   Crystal structure of cas9 in complex with guide RNA and target        DNA. Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S.,        Shehata, S I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O.        Cell February 27, 156(5):935-49 (2014);    -   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian        cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon        D B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch        R., Zhang F., Sharp P A. Nat Biotechnol. April 20. doi:        10.1038/nbt.2889 (2014);    -   CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.        Platt R J, Chen S, Zhou Y, Yim M J, Swiech L, Kempton H R,        Dahlman J E, Parnas O, Eisenhaure T M, Jovanovic M, Graham D B,        Jhunjhunwala S, Heidenreich M, Xavier R J, Langer R, Anderson D        G, Hacohen N, Regev A, Feng G, Sharp P A, Zhang F. Cell 159(2):        440-455 DOI: 10.1016/j.cell.2014.09.014(2014);    -   Development and Applications of CRISPR-Cas9 for Genome        Engineering, Hsu P D, Lander E S, Zhang F., Cell. June 5;        157(6):1262-78 (2014).    -   Genetic screens in human cells using the CRISPR-Cas9 system,        Wang T, Wei J J, Sabatini D M, Lander E S., Science. January 3;        343(6166): 80-84. doi:10.1126/science.1246981 (2014);    -   Rational design of highly active sgRNAs for CRISPR-Cas9-mediated        gene inactivation, Doench J G, Hartenian E, Graham D B, Tothova        Z, Hegde M, Smith I, Sullender M, Ebert B L, Xavier R J, Root D        E., (published online 3 Sep. 2014) Nat Biotechnol. December;        32(12):1262-7 (2014);    -   In vivo interrogation of gene function in the mammalian brain        using CRISPR-Cas9, Swiech L, Heidenreich M, Banerjee A, Habib N,        Li Y, Trombetta J, Sur M, Zhang F., (published online 19        Oct. 2014) Nat Biotechnol. January; 33(1):102-6 (2015);    -   Genome-scale transcriptional activation by an engineered        CRISPR-Cas9 complex, Konermann S, Brigham M D, Trevino A E,        Joung J, Abudayyeh O O, Barcena C, Hsu P D, Habib N, Gootenberg        J S, Nishimasu H, Nureki O, Zhang F., Nature. January 29;        517(7536):583-8 (2015).    -   A split-Cas9 architecture for inducible genome editing and        transcription modulation, Zetsche B, Volz S E, Zhang F.,        (published online 2 Feb. 2015) Nat Biotechnol. February;        33(2):139-42 (2015);    -   Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and        Metastasis, Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi        X, Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F,        Sharp P A. Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen        in mouse), and    -   In vivo genome editing using Staphylococcus aureus Cas9, Ran F        A, Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche        B, Shalem O, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang        F., (published online 1 Apr. 2015), Nature. April 9; 520(7546):        186-91 (2015).    -   Shalem et al., “High-throughput functional genomics using        CRISPR-Cas9,” Nature Reviews Genetics 16, 299-311 (May 2015).    -   Xu et al., “Sequence determinants of improved CRISPR sgRNA        design,” Genome Research 25, 1147-1157 (August 2015).    -   Parnas et al., “A Genome-wide CRISPR Screen in Primary Immune        Cells to Dissect Regulatory Networks,” Cell 162, 675-686 (Jul.        30, 2015).    -   Ramanan et al., CRISPR-Cas9 cleavage of viral DNA efficiently        suppresses hepatitis B virus,” Scientific Reports 5:10833. doi:        10.1038/srep10833 (Jun. 2, 2015)    -   Nishimasu et al., Crystal Structure of Staphylococcus aureus        Cas9,” Cell 162, 1113-1126 (Aug. 27, 2015)    -   BCL11A enhancer dissection by Cas9-mediated in situ saturating        mutagenesis, Canver et al., Nature 527(7577):192-7 (Nov.        12, 2015) doi: 10.1038/nature15521. Epub 2015 Sep. 16.    -   Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas        System, Zetsche et al., Cell 163, 759-71 (Sep. 25, 2015).    -   Discovery and Functional Characterization of Diverse Class 2        CRISPR-Cas Systems, Shmakov et al., Molecular Cell, 60(3),        385-397 doi: 10.1016/j.molcel.2015.10.008 Epub Oct. 22, 2015.    -   Rationally engineered Cas9 nucleases with improved specificity,        Slaymaker et al., Science 2016 Jan. 1 351(6268): 84-88 doi:        10.1126/science.aad5227. Epub 2015 Dec. 1.    -   Gao et al, “Engineered Cpf1 Enzymes with Altered PAM        Specificities,” bioRxiv 091611; doi:        http://dx.doi.org/10.1101/091611 (Dec. 4, 2016).    -   Cox et al., “RNA editing with CRISPR-Cas13,” Science. 2017 Nov.        24; 358(6366):1019-1027. doi: 10.1126/science.aaq0180. Epub 2017        Oct. 25.

each of which is incorporated herein by reference, may be considered inthe practice of the instant invention, and discussed briefly below:

-   -   Cong et al. engineered type II CRISPR-Cas systems for use in        eukaryotic cells based on both Streptococcus thermophilus Cas9        and also Streptococcus pyogenes Cas9 and demonstrated that Cas9        nucleases can be directed by short RNAs to induce precise        cleavage of DNA in human and mouse cells. Their study further        showed that Cas9 as converted into a nicking enzyme can be used        to facilitate homology-directed repair in eukaryotic cells with        minimal mutagenic activity. Additionally, their study        demonstrated that multiple guide sequences can be encoded into a        single CRISPR array to enable simultaneous editing of several at        endogenous genomic loci sites within the mammalian genome,        demonstrating easy programmability and wide applicability of the        RNA-guided nuclease technology. This ability to use RNA to        program sequence specific DNA cleavage in cells defined a new        class of genome engineering tools. These studies further showed        that other CRISPR loci are likely to be transplantable into        mammalian cells and can also mediate mammalian genome cleavage.        Importantly, it can be envisaged that several aspects of the        CRISPR-Cas system can be further improved to increase its        efficiency and versatility.    -   Jiang et al. used the clustered, regularly interspaced, short        palindromic repeats (CRISPR)-associated Cas9 endonuclease        complexed with dual-RNAs to introduce precise mutations in the        genomes of Streptococcus pneumoniae and Escherichia coli. The        approach relied on dual-RNA:Cas9-directed cleavage at the        targeted genomic site to kill unmutated cells and circumvents        the need for selectable markers or counter-selection systems.        The study reported reprogramming dual-RNA:Cas9 specificity by        changing the sequence of short CRISPR RNA (crRNA) to make        single- and multinucleotide changes carried on editing        templates. The study showed that simultaneous use of two crRNAs        enabled multiplex mutagenesis. Furthermore, when the approach        was used in combination with recombineering, in S. pneumoniae,        nearly 100% of cells that were recovered using the described        approach contained the desired mutation, and in E. coli, 65%        that were recovered contained the mutation.    -   Wang et al. (2013) used the CRISPR-Cas system for the one-step        generation of mice carrying mutations in multiple genes which        were traditionally generated in multiple steps by sequential        recombination in embryonic stem cells and/or time-consuming        intercrossing of mice with a single mutation. The CRISPR-Cas        system will greatly accelerate the in vivo study of functionally        redundant genes and of epistatic gene interactions.    -   Konermann et al. (2013) addressed the need in the art for        versatile and robust technologies that enable optical and        chemical modulation of DNA-binding domains based CRISPR Cas9        enzyme and also Transcriptional Activator Like Effectors    -   Ran et al. (2013-A) described an approach that combined a Cas9        nickase mutant with paired guide RNAs to introduce targeted        double-strand breaks. This addresses the issue of the Cas9        nuclease from the microbial CRISPR-Cas system being targeted to        specific genomic loci by a guide sequence, which can tolerate        certain mismatches to the DNA target and thereby promote        undesired off-target mutagenesis. Because individual nicks in        the genome are repaired with high fidelity, simultaneous nicking        via appropriately offset guide RNAs is required for        double-stranded breaks and extends the number of specifically        recognized bases for target cleavage. The authors demonstrated        that using paired nicking can reduce off-target activity by 50-        to 1,500-fold in cell lines and to facilitate gene knockout in        mouse zygotes without sacrificing on-target cleavage efficiency.        This versatile strategy enables a wide variety of genome editing        applications that require high specificity.    -   Hsu et al. (2013) characterized SpCas9 targeting specificity in        human cells to inform the selection of target sites and avoid        off-target effects. The study evaluated >700 guide RNA variants        and SpCas9-induced indel mutation levels at >100 predicted        genomic off-target loci in 293T and 293FT cells. The authors        that SpCas9 tolerates mismatches between guide RNA and target        DNA at different positions in a sequence-dependent manner,        sensitive to the number, position and distribution of        mismatches. The authors further showed that SpCas9-mediated        cleavage is unaffected by DNA methylation and that the dosage of        SpCas9 and guide RNA can be titrated to minimize off-target        modification. Additionally, to facilitate mammalian genome        engineering applications, the authors reported providing a        web-based software tool to guide the selection and validation of        target sequences as well as off-target analyses.    -   Ran et al. (2013-B) described a set of tools for Cas9-mediated        genome editing via non-homologous end joining (NHEJ) or        homology-directed repair (HDR) in mammalian cells, as well as        generation of modified cell lines for downstream functional        studies. To minimize off-target cleavage, the authors further        described a double-nicking strategy using the Cas9 nickase        mutant with paired guide RNAs. The protocol provided by the        authors experimentally derived guidelines for the selection of        target sites, evaluation of cleavage efficiency and analysis of        off-target activity. The studies showed that beginning with        target design, gene modifications can be achieved within as        little as 1-2 weeks, and modified clonal cell lines can be        derived within 2-3 weeks.    -   Shalem et al. described a new way to interrogate gene function        on a genome-wide scale. Their studies showed that delivery of a        genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted        18,080 genes with 64,751 unique guide sequences enabled both        negative and positive selection screening in human cells. First,        the authors showed use of the GeCKO library to identify genes        essential for cell viability in cancer and pluripotent stem        cells. Next, in a melanoma model, the authors screened for genes        whose loss is involved in resistance to vemurafenib, a        therapeutic that inhibits mutant protein kinase BRAF. Their        studies showed that the highest-ranking candidates included        previously validated genes NF1 and MED12 as well as novel hits        NF2, CUL3, TADA2B, and TADA1. The authors observed a high level        of consistency between independent guide RNAs targeting the same        gene and a high rate of hit confirmation, and thus demonstrated        the promise of genome-scale screening with Cas9.    -   Nishimasu et al. reported the crystal structure of Streptococcus        pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°        resolution. The structure revealed a bilobed architecture        composed of target recognition and nuclease lobes, accommodating        the sgRNA:DNA heteroduplex in a positively charged groove at        their interface. Whereas the recognition lobe is essential for        binding sgRNA and DNA, the nuclease lobe contains the HNH and        RuvC nuclease domains, which are properly positioned for        cleavage of the complementary and non-complementary strands of        the target DNA, respectively. The nuclease lobe also contains a        carboxyl-terminal domain responsible for the interaction with        the protospacer adjacent motif (PAM). This high-resolution        structure and accompanying functional analyses have revealed the        molecular mechanism of RNA-guided DNA targeting by Cas9, thus        paving the way for the rational design of new, versatile        genome-editing technologies.    -   Wu et al. mapped genome-wide binding sites of a catalytically        inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with        single guide RNAs (sgRNAs) in mouse embryonic stem cells        (mESCs). The authors showed that each of the four sgRNAs tested        targets dCas9 to between tens and thousands of genomic sites,        frequently characterized by a 5-nucleotide seed region in the        sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin        inaccessibility decreases dCas9 binding to other sites with        matching seed sequences; thus 70% of off-target sites are        associated with genes. The authors showed that targeted        sequencing of 295 dCas9 binding sites in mESCs transfected with        catalytically active Cas9 identified only one site mutated above        background levels. The authors proposed a two-state model for        Cas9 binding and cleavage, in which a seed match triggers        binding but extensive pairing with target DNA is required for        cleavage.    -   Platt et al. established a Cre-dependent Cas9 knockin mouse. The        authors demonstrated in vivo as well as ex vivo genome editing        using adeno-associated virus (AAV)-, lentivirus-, or        particle-mediated delivery of guide RNA in neurons, immune        cells, and endothelial cells.    -   Hsu et al. (2014) is a review article that discusses generally        CRISPR-Cas9 history from yogurt to genome editing, including        genetic screening of cells.    -   Wang et al. (2014) relates to a pooled, loss-of-function genetic        screening approach suitable for both positive and negative        selection that uses a genome-scale lentiviral single guide RNA        (sgRNA) library.    -   Doench et al. created a pool of sgRNAs, tiling across all        possible target sites of a panel of six endogenous mouse and        three endogenous human genes and quantitatively assessed their        ability to produce null alleles of their target gene by antibody        staining and flow cytometry. The authors showed that        optimization of the PAM improved activity and also provided an        on-line tool for designing sgRNAs.    -   Swiech et al. demonstrate that AAV-mediated SpCas9 genome        editing can enable reverse genetic studies of gene function in        the brain.    -   Konermann et al. (2015) discusses the ability to attach multiple        effector domains, e.g., transcriptional activator, functional        and epigenomic regulators at appropriate positions on the guide        such as stem or tetraloop with and without linkers.    -   Zetsche et al. demonstrates that the Cas9 enzyme can be split        into two and hence the assembly of Cas9 for activation can be        controlled.    -   Chen et al. relates to multiplex screening by demonstrating that        a genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes        regulating lung metastasis.    -   Ran et al. (2015) relates to SaCas9 and its ability to edit        genomes and demonstrates that one cannot extrapolate from        biochemical assays.    -   Shalem et al. (2015) described ways in which catalytically        inactive Cas9 (dCas9) fusions are used to synthetically repress        (CRISPRi) or activate (CRISPRa) expression, showing. advances        using Cas9 for genome-scale screens, including arrayed and        pooled screens, knockout approaches that inactivate genomic loci        and strategies that modulate transcriptional activity.    -   Xu et al. (2015) assessed the DNA sequence features that        contribute to single guide RNA (sgRNA) efficiency in        CRISPR-based screens. The authors explored efficiency of        CRISPR-Cas9 knockout and nucleotide preference at the cleavage        site. The authors also found that the sequence preference for        CRISPRi/a is substantially different from that for CRISPR-Cas9        knockout.    -   Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9        libraries into dendritic cells (DCs) to identify genes that        control the induction of tumor necrosis factor (Tnf) by        bacterial lipopolysaccharide (LPS). Known regulators of Tlr4        signaling and previously unknown candidates were identified and        classified into three functional modules with distinct effects        on the canonical responses to LPS.    -   Ramanan et al (2015) demonstrated cleavage of viral episomal DNA        (cccDNA) in infected cells. The HBV genome exists in the nuclei        of infected hepatocytes as a 3.2 kb double-stranded episomal DNA        species called covalently closed circular DNA (cccDNA), which is        a key component in the HBV life cycle whose replication is not        inhibited by current therapies. The authors showed that sgRNAs        specifically targeting highly conserved regions of HBV robustly        suppresses viral replication and depleted cccDNA.    -   Nishimasu et al. (2015) reported the crystal structures of        SaCas9 in complex with a single guide RNA (sgRNA) and its        double-stranded DNA targets, containing the 5′-TTGAAT-3′ PAM and        the 5′-TTGGGT-3′ PAM. A structural comparison of SaCas9 with        SpCas9 highlighted both structural conservation and divergence,        explaining their distinct PAM specificities and orthologous        sgRNA recognition.    -   Canver et al. (2015) demonstrated a CRISPR-Cas9-based functional        investigation of non-coding genomic elements. The authors we        developed pooled CRISPR-Cas9 guide RNA libraries to perform in        situ saturating mutagenesis of the human and mouse BCL11A        enhancers which revealed critical features of the enhancers.    -   Zetsche et al. (2015) reported characterization of Cpf1, a class        2 CRISPR nuclease from Francisella novicida U112 having features        distinct from Cas9. Cpf1 is a single RNA-guided endonuclease        lacking tracrRNA, utilizes a T-rich protospacer-adjacent motif,        and cleaves DNA via a staggered DNA double-stranded break.    -   Shmakov et al. (2015) reported three distinct Class 2 CRISPR-Cas        systems. Two system CRISPR enzymes (C2c1 and C2c3) contain        RuvC-like endonuclease domains distantly related to Cpf1. Unlike        Cpf1, C2c1 depends on both crRNA and tracrRNA for DNA cleavage.        The third enzyme (C2c2) contains two predicted HEPN RNase        domains and is tracrRNA independent.    -   Slaymaker et al (2016) reported the use of structure-guided        protein engineering to improve the specificity of Streptococcus        pyogenes Cas9 (SpCas9). The authors developed “enhanced        specificity” SpCas9 (eSpCas9) variants which maintained robust        on-target cleavage with reduced off-target effects.    -   Cox et al., (2017) reported the use of catalytically inactive        Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity        by ADAR2 (adenosine deaminase acting on RNA type 2) to        transcripts in mammalian cells. The system, referred to as RNA        Editing for Programmable A to I Replacement (REPAIR), has no        strict sequence constraints and can be used to edit full-length        transcripts. The authors further engineered the system to create        a high-specificity variant and minimized the system to        facilitate viral delivery.

The methods and tools provided herein are may be designed for use withor Cas13, a type II nuclease that does not make use of tracrRNA.Orthologs of Cas13 have been identified in different bacterial speciesas described herein. Further type II nucleases with similar propertiescan be identified using methods described in the art (Shmakov et al.2015, 60:385-397; Abudayeh et al. 2016, Science, 5; 353(6299)). Inparticular embodiments, such methods for identifying novel CRISPReffector proteins may comprise the steps of selecting sequences from thedatabase encoding a seed which identifies the presence of a CRISPR Caslocus, identifying loci located within 10 kb of the seed comprising OpenReading Frames (ORFs) in the selected sequences, selecting therefromloci comprising ORFs of which only a single ORF encodes a novel CRISPReffector having greater than 700 amino acids and no more than 90%homology to a known CRISPR effector. In particular embodiments, the seedis a protein that is common to the CRISPR-Cas system, such as Cas1. Infurther embodiments, the CRISPR array is used as a seed to identify neweffector proteins.

Also, “Dimeric CRISPR RNA-guided FokI nucleases for highly specificgenome editing”, Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter,Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin,Martin J. Aryee, J. Keith Joung Nature Biotechnology 32(6): 569-77(2014), relates to dimeric RNA-guided FokI Nucleases that recognizeextended sequences and can edit endogenous genes with high efficienciesin human cells.

With respect to general information on CRISPR/Cas Systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles, and making and using thereof,including as to amounts and formulations, as well asCRISPR-Cas-expressing eukaryotic cells, CRISPR-Cas expressingeukaryotes, such as a mouse, reference is made to: U.S. Pat. Nos.8,999,641, 8,993,233, 8,697,359, 8,771,945, 8,795,965, 8,865,406,8,871,445, 8,889,356, 8,889,418, 8,895,308, 8,906,616, 8,932,814, and8,945,839; US Patent Publications US 2014-0310830 (U.S. application Ser.No. 14/105,031), US 2014-0287938 A1 (U.S. application Ser. No.14/213,991), US 2014-0273234 A1 (U.S. application Ser. No. 14/293,674),US2014-0273232 A1 (U.S. application Ser. No. 14/290,575), US2014-0273231 (U.S. application Ser. No. 14/259,420), US 2014-0256046 A1(U.S. application Ser. No. 14/226,274), US 2014-0248702 A1 (U.S.application Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. applicationSer. No. 14/222,930), US 2014-0242699 A1 (U.S. application Ser. No.14/183,512), US 2014-0242664 A1 (U.S. application Ser. No. 14/104,990),US 2014-0234972 A1 (U.S. application Ser. No. 14/183,471), US2014-0227787 A1 (U.S. application Ser. No. 14/256,912), US 2014-0189896A1 (U.S. application Ser. No. 14/105,035), US 2014-0186958 (U.S.application Ser. No. 14/105,017), US 2014-0186919 A1 (U.S. applicationSer. No. 14/104,977), US 2014-0186843 A1 (U.S. application Ser. No.14/104,900), US 2014-0179770 A1 (U.S. application Ser. No. 14/104,837)and US 2014-0179006 A1 (U.S. application Ser. No. 14/183,486), US2014-0170753 (U.S. application Ser. No. 14/183,429); US 2015-0184139(U.S. application Ser. No. 14/324,960); Ser. No. 14/054,414 EuropeanPatent Applications EP 2 771 468 (EP13818570.7), EP 2 764 103(EP13824232.6), and EP 2 784 162 (EP14170383.5); and PCT PatentPublications WO2014/093661 (PCT/US2013/074743), WO2014/093694(PCT/US2013/074790), WO2014/093595 (PCT/US2013/074611), WO2014/093718(PCT/US2013/074825), WO2014/093709 (PCT/US2013/074812), WO2014/093622(PCT/US2013/074667), WO2014/093635 (PCT/US2013/074691), WO2014/093655(PCT/US2013/074736), WO2014/093712 (PCT/US2013/074819), WO2014/093701(PCT/US2013/074800), WO2014/018423 (PCT/US2013/051418), WO2014/204723(PCT/US2014/041790), WO2014/204724 (PCT/US2014/041800), WO2014/204725(PCT/US2014/041803), WO2014/204726 (PCT/US2014/041804), WO2014/204727(PCT/US2014/041806), WO2014/204728 (PCT/US2014/041808), WO2014/204729(PCT/US2014/041809), WO2015/089351 (PCT/US2014/069897), WO2015/089354(PCT/US2014/069902), WO2015/089364 (PCT/US2014/069925), WO2015/089427(PCT/US2014/070068), WO2015/089462 (PCT/US2014/070127), WO2015/089419(PCT/US2014/070057), WO2015/089465 (PCT/US2014/070135), WO2015/089486(PCT/US2014/070175), WO2015/058052 (PCT/US2014/061077), WO2015/070083(PCT/US2014/064663), WO2015/089354 (PCT/US2014/069902), WO2015/089351(PCT/US2014/069897), WO2015/089364 (PCT/US2014/069925), WO2015/089427(PCT/US2014/070068), WO2015/089473 (PCT/US2014/070152), WO2015/089486(PCT/US2014/070175), WO2016/049258 (PCT/US2015/051830), WO2016/094867(PCT/US2015/065385), WO2016/094872 (PCT/US2015/065393), WO2016/094874(PCT/US2015/065396), WO2016/106244 (PCT/US2015/067177).

Mention is also made of U.S. application 62/180,709, 17 Jun. 2015,PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,455, filed, 12Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/096,708,24 Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. applications62/091,462, 12 Dec. 2014, 62/096,324, 23 Dec. 2014, 62/180,681, 17 Jun.2015, and 62/237,496, 5 Oct. 2015, DEAD GUIDES FOR CRISPR TRANSCRIPTIONFACTORS; U.S. application 62/091,456, 12 Dec. 2014 and 62/180,692, 17Jun. 2015, ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS;U.S. application 62/091,461, 12 Dec. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOMEEDITING AS TO HEMATOPOETIC STEM CELLS (HSCs); U.S. application62/094,903, 19 Dec. 2014, UNBIASED IDENTIFICATION OF DOUBLE-STRANDBREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURESEQUENCING; U.S. application 62/096,761, 24 Dec. 2014, ENGINEERING OFSYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCEMANIPULATION; U.S. application 62/098,059, 30 Dec. 2014, 62/181,641, 18Jun. 2015, and 62/181,667, 18 Jun. 2015, RNA-TARGETING SYSTEM; U.S.application 62/096,656, 24 Dec. 2014 and 62/181,151, 17 Jun. 2015,CRISPR HAVING OR ASSOCIATED WITH DESTABILIZATION DOMAINS; U.S.application 62/096,697, 24 Dec. 2014, CRISPR HAVING OR ASSOCIATED WITHAAV; U.S. application 62/098,158, 30 Dec. 2014, ENGINEERED CRISPRCOMPLEX INSERTIONAL TARGETING SYSTEMS; U.S. application 62/151,052, 22Apr. 2015, CELLULAR TARGETING FOR EXTRACELLULAR EXOSOMAL REPORTING; U.S.application 62/054,490, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETINGDISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS; U.S.application 61/939,154, 12-F

EB-14, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITHOPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/055,484, 25Sep. 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONWITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application62/087,537, 4 Dec. 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCEMANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/054,651, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELINGCOMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S. application62/067,886, 23 Oct. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OFMULTIPLE CANCER MUTATIONS IN VIVO; U.S. applications 62/054,675, 24 Sep.2014 and 62/181,002, 17 Jun. 2015, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN NEURONALCELLS/TISSUES; U.S. application 62/054,528, 24 Sep. 2014, DELIVERY, USEAND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONSIN IMMUNE DISEASES OR DISORDERS; U.S. application 62/055,454, 25 Sep.2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING CELLPENETRATION PEPTIDES (CPP); U.S. application 62/055,460, 25 Sep. 2014,MULTIFUNCTIONAL-CRISPR COMPLEXES AND/OR OPTIMIZED ENZYME LINKEDFUNCTIONAL-CRISPR COMPLEXES; U.S. application 62/087,475, 4 Dec. 2014and 62/181,690, 18 Jun. 2015, FUNCTIONAL SCREENING WITH OPTIMIZEDFUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/055,487, 25 Sep.2014, FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;U.S. application 62/087,546, 4 Dec. 2014 and 62/181,687, 18 Jun. 2015,MULTIFUNCTIONAL CRISPR COMPLEXES AND/OR OPTIMIZED ENZYME LINKEDFUNCTIONAL-CRISPR COMPLEXES; and U.S. application 62/098,285, 30 Dec.2014, CRISPR MEDIATED IN VIVO MODELING AND GENETIC SCREENING OF TUMORGROWTH AND METASTASIS.

Mention is made of U.S. applications 62/181,659, 18 Jun. 2015 and62/207,318, 19 Aug. 2015, ENGINEERING AND OPTIMIZATION OF SYSTEMS,METHODS, ENZYME AND GUIDE SCAFFOLDS OF CAS9 ORTHOLOGS AND VARIANTS FORSEQUENCE MANIPULATION. Mention is made of U.S. applications 62/181,663,18 Jun. 2015 and 62/245,264, 22 Oct. 2015, NOVEL CRISPR ENZYMES ANDSYSTEMS, U.S. applications 62/181,675, 18 Jun. 2015, 62/285,349, 22 Oct.2015, 62/296,522, 17 Feb. 2016, and 62/320,231, 8 Apr. 2016, NOVELCRISPR ENZYMES AND SYSTEMS, U.S. application 62/232,067, 24 Sep. 2015,U.S. application Ser. No. 14/975,085, 18 Dec. 2015, European applicationNo. 16150428.7, U.S. application 62/205,733, 16 Aug. 2015, U.S.application 62/201,542, 5 Aug. 2015, U.S. application 62/193,507, 16Jul. 2015, and U.S. application 62/181,739, 18 Jun. 2015, each entitledNOVEL CRISPR ENZYMES AND SYSTEMS and of U.S. application 62/245,270, 22Oct. 2015, NOVEL CRISPR ENZYMES AND SYSTEMS. Mention is also made ofU.S. application 61/939,256, 12 Feb. 2014, and WO 2015/089473(PCT/US2014/070152), 12 Dec. 2014, each entitled ENGINEERING OF SYSTEMS,METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEW ARCHITECTURES FORSEQUENCE MANIPULATION. Mention is also made of PCT/US2015/045504, 15Aug. 2015, U.S. application 62/180,699, 17 Jun. 2015, and U.S.application 62/038,358, 17 Aug. 2014, each entitled GENOME EDITING USINGCAS9 NICKASES.

Each of these patents, patent publications, and applications, and alldocuments cited therein or during their prosecution (“appln citeddocuments”) and all documents cited or referenced in the appln citeddocuments, together with any instructions, descriptions, productspecifications, and product sheets for any products mentioned therein orin any document therein and incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. All documents (e.g., these patents, patent publicationsand applications and the appln cited documents) are incorporated hereinby reference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

In particular embodiments, pre-complexed guide RNA and CRISPR effectorprotein, (optionally, adenosine deaminase fused to a CRISPR protein oran adaptor) are delivered as a ribonucleoprotein (RNP). RNPs have theadvantage that they lead to rapid editing effects even more so than theRNA method because this process avoids the need for transcription. Animportant advantage is that both RNP delivery is transient, reducingoff-target effects and toxicity issues. Efficient genome editing indifferent cell types has been observed by Kim et al. (2014, Genome Res.24(6):1012-9), Paix et al. (2015, Genetics 204(1):47-54), Chu et al.(2016, BMC Biotechnol. 16:4), and Wang et al. (2013, Cell. 9; 153 (4):910-8).

In particular embodiments, the ribonucleoprotein is delivered by way ofa polypeptide-based shuttle agent as described in WO2016161516.WO2016161516 describes efficient transduction of polypeptide cargosusing synthetic peptides comprising an endosome leakage domain (ELD)operably linked to a cell penetrating domain (CPD), to a histidine-richdomain and a CPD. Similarly these polypeptides can be used for thedelivery of CRISPR-effector based RNPs in eukaryotic cells.

Tale Systems

As disclosed herein editing can be made by way of the transcriptionactivator-like effector nucleases (TALENs) system. Transcriptionactivator-like effectors (TALEs) can be engineered to bind practicallyany desired DNA sequence. Exemplary methods of genome editing using theTALEN system can be found for example in Cermak T. Doyle E L. ChristianM. Wang L. Zhang Y. Schmidt C, et al. Efficient design and assembly ofcustom TALEN and other TAL effector-based constructs for DNA targeting.Nucleic Acids Res. 2011; 39:e82; Zhang F. Cong L. Lodato S. Kosuri S.Church G M. Arlotta P Efficient construction of sequence-specific TALeffectors for modulating mammalian transcription. Nat Biotechnol. 2011;29:149-153 and U.S. Pat. Nos. 8,450,471, 8,440,431 and 8,440,432, all ofwhich are specifically incorporated by reference.

In advantageous embodiments of the invention, the methods providedherein use isolated, non-naturally occurring, recombinant or engineeredDNA binding proteins that comprise TALE monomers as a part of theirorganizational structure that enable the targeting of nucleic acidsequences with improved efficiency and expanded specificity.

Naturally occurring TALEs or “wild type TALEs” are nucleic acid bindingproteins secreted by numerous species of proteobacteria. TALEpolypeptides contain a nucleic acid binding domain composed of tandemrepeats of highly conserved monomer polypeptides that are predominantly33, 34 or 35 amino acids in length and that differ from each othermainly in amino acid positions 12 and 13. In advantageous embodimentsthe nucleic acid is DNA. As used herein, the term “polypeptidemonomers”, or “TALE monomers” will be used to refer to the highlyconserved repetitive polypeptide sequences within the TALE nucleic acidbinding domain and the term “repeat variable di-residues” or “RVD” willbe used to refer to the highly variable amino acids at positions 12 and13 of the polypeptide monomers. As provided throughout the disclosure,the amino acid residues of the RVD are depicted using the IUPAC singleletter code for amino acids. A general representation of a TALE monomerwhich is comprised within the DNA binding domain isX1-11-(X12X13)-X14-33 or 34 or 35, where the subscript indicates theamino acid position and X represents any amino acid. X12X13 indicate theRVDs. In some polypeptide monomers, the variable amino acid at position13 is missing or absent and in such polypeptide monomers, the RVDconsists of a single amino acid. In such cases the RVD may bealternatively represented as X*, where X represents X12 and (*)indicates that X13 is absent. The DNA binding domain comprises severalrepeats of TALE monomers and this may be represented as(X1-11-(X12X13)-X14-33 or 34 or 35)z, where in an advantageousembodiment, z is at least 5 to 40. In a further advantageous embodiment,z is at least 10 to 26.

The TALE monomers have a nucleotide binding affinity that is determinedby the identity of the amino acids in its RVD. For example, polypeptidemonomers with an RVD of NI preferentially bind to adenine (A),polypeptide monomers with an RVD of NG preferentially bind to thymine(T), polypeptide monomers with an RVD of HD preferentially bind tocytosine (C) and polypeptide monomers with an RVD of NN preferentiallybind to both adenine (A) and guanine (G). In yet another embodiment ofthe invention, polypeptide monomers with an RVD of IG preferentiallybind to T. Thus, the number and order of the polypeptide monomer repeatsin the nucleic acid binding domain of a TALE determines its nucleic acidtarget specificity. In still further embodiments of the invention,polypeptide monomers with an RVD of NS recognize all four base pairs andmay bind to A, T, G or C. The structure and function of TALEs is furtherdescribed in, for example, Moscou et al., Science 326:1501 (2009); Bochet al., Science 326:1509-1512 (2009); and Zhang et al., NatureBiotechnology 29:149-153 (2011), each of which is incorporated byreference in its entirety.

The TALE polypeptides used in methods of the invention are isolated,non-naturally occurring, recombinant or engineered nucleic acid-bindingproteins that have nucleic acid or DNA binding regions containingpolypeptide monomer repeats that are designed to target specific nucleicacid sequences.

As described herein, polypeptide monomers having an RVD of HN or NHpreferentially bind to guanine and thereby allow the generation of TALEpolypeptides with high binding specificity for guanine containing targetnucleic acid sequences. In a preferred embodiment of the invention,polypeptide monomers having RVDs RN, NN, NK, SN, NH, KN, HN, NQ, HH, RG,KH, RH and SS preferentially bind to guanine. In a much moreadvantageous embodiment of the invention, polypeptide monomers havingRVDs RN, NK, NQ, HH, KH, RH, SS and SN preferentially bind to guanineand thereby allow the generation of TALE polypeptides with high bindingspecificity for guanine containing target nucleic acid sequences. In aneven more advantageous embodiment of the invention, polypeptide monomershaving RVDs HH, KH, NH, NK, NQ, RH, RN and SS preferentially bind toguanine and thereby allow the generation of TALE polypeptides with highbinding specificity for guanine containing target nucleic acidsequences. In a further advantageous embodiment, the RVDs that have highbinding specificity for guanine are RN, NH RH and KH. Furthermore,polypeptide monomers having an RVD of NV preferentially bind to adenineand guanine. In more preferred embodiments of the invention, polypeptidemonomers having RVDs of H*, HA, KA, N*, NA, NC, NS, RA, and S* bind toadenine, guanine, cytosine and thymine with comparable affinity.

The predetermined N-terminal to C-terminal order of the one or morepolypeptide monomers of the nucleic acid or DNA binding domaindetermines the corresponding predetermined target nucleic acid sequenceto which the TALE polypeptides will bind. As used herein the polypeptidemonomers and at least one or more half polypeptide monomers are“specifically ordered to target” the genomic locus or gene of interest.In plant genomes, the natural TALE-binding sites always begin with athymine (T), which may be specified by a cryptic signal within thenon-repetitive N-terminus of the TALE polypeptide; in some cases thisregion may be referred to as repeat 0. In animal genomes, TALE bindingsites do not necessarily have to begin with a thymine (T) and TALEpolypeptides may target DNA sequences that begin with T, A, G or C. Thetandem repeat of TALE monomers always ends with a half-length repeat ora stretch of sequence that may share identity with only the first 20amino acids of a repetitive full length TALE monomer and this halfrepeat may be referred to as a half-monomer (FIG. 8), which is includedin the term “TALE monomer”. Therefore, it follows that the length of thenucleic acid or DNA being targeted is equal to the number of fullpolypeptide monomers plus two.

As described in Zhang et al., Nature Biotechnology 29:149-153 (2011),TALE polypeptide binding efficiency may be increased by including aminoacid sequences from the “capping regions” that are directly N-terminalor C-terminal of the DNA binding region of naturally occurring TALEsinto the engineered TALEs at positions N-terminal or C-terminal of theengineered TALE DNA binding region. Thus, in certain embodiments, theTALE polypeptides described herein further comprise an N-terminalcapping region and/or a C-terminal capping region.

An exemplary amino acid sequence of a N-terminal capping region is:

(SEQ. I.D. No: 4) MDPIRSRTPSPARELLSGPQPDGVQPTADRGVSPPAGGPLDGLPARRTMSRTRLPSPPAPSPAFSADS FSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLRAADAPPPTMRVAVTAARPPRAKPA PRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALG TVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAV EAVHAWRNALTGAPLNAn exemplary amino acid sequence of a C-terminal capping region is:

(SEQ. I.D. No. 5) RPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHR VADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDR ILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMHEGDQTRAS

As used herein the predetermined “N-terminus” to “C terminus”orientation of the N-terminal capping region, the DNA binding domaincomprising the repeat TALE monomers and the C-terminal capping regionprovide structural basis for the organization of different domains inthe d-TALEs or polypeptides of the invention.

The entire N-terminal and/or C-terminal capping regions are notnecessary to enhance the binding activity of the DNA binding region.Therefore, in certain embodiments, fragments of the N-terminal and/orC-terminal capping regions are included in the TALE polypeptidesdescribed herein.

In certain embodiments, the TALE polypeptides described herein contain aN-terminal capping region fragment that included at least 10, 20, 30,40, 50, 54, 60, 70, 80, 87, 90, 94, 100, 102, 110, 117, 120, 130, 140,147, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or 270amino acids of an N-terminal capping region. In certain embodiments, theN-terminal capping region fragment amino acids are of the C-terminus(the DNA-binding region proximal end) of an N-terminal capping region.As described in Zhang et al., Nature Biotechnology 29:149-153 (2011),N-terminal capping region fragments that include the C-terminal 240amino acids enhance binding activity equal to the full length cappingregion, while fragments that include the C-terminal 147 amino acidsretain greater than 80% of the efficacy of the full length cappingregion, and fragments that include the C-terminal 117 amino acids retaingreater than 50% of the activity of the full-length capping region.

In some embodiments, the TALE polypeptides described herein contain aC-terminal capping region fragment that included at least 6, 10, 20, 30,37, 40, 50, 60, 68, 70, 80, 90, 100, 110, 120, 127, 130, 140, 150, 155,160, 170, 180 amino acids of a C-terminal capping region. In certainembodiments, the C-terminal capping region fragment amino acids are ofthe N-terminus (the DNA-binding region proximal end) of a C-terminalcapping region. As described in Zhang et al., Nature Biotechnology29:149-153 (2011), C-terminal capping region fragments that include theC-terminal 68 amino acids enhance binding activity equal to the fulllength capping region, while fragments that include the C-terminal 20amino acids retain greater than 50% of the efficacy of the full lengthcapping region.

In certain embodiments, the capping regions of the TALE polypeptidesdescribed herein do not need to have identical sequences to the cappingregion sequences provided herein. Thus, in some embodiments, the cappingregion of the TALE polypeptides described herein have sequences that areat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical or share identity to the capping region aminoacid sequences provided herein. Sequence identity is related to sequencehomology. Homology comparisons may be conducted by eye, or more usually,with the aid of readily available sequence comparison programs. Thesecommercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences. In some preferred embodiments, the capping region of the TALEpolypeptides described herein have sequences that are at least 95%identical or share identity to the capping region amino acid sequencesprovided herein.

Sequence homologies may be generated by any of a number of computerprograms known in the art, which include but are not limited to BLAST orFASTA. Suitable computer program for carrying out alignments like theGCG Wisconsin Bestfit package may also be used. Once the software hasproduced an optimal alignment, it is possible to calculate % homology,preferably % sequence identity. The software typically does this as partof the sequence comparison and generates a numerical result.

In advantageous embodiments described herein, the TALE polypeptides ofthe invention include a nucleic acid binding domain linked to the one ormore effector domains. The terms “effector domain” or “regulatory andfunctional domain” refer to a polypeptide sequence that has an activityother than binding to the nucleic acid sequence recognized by thenucleic acid binding domain. By combining a nucleic acid binding domainwith one or more effector domains, the polypeptides of the invention maybe used to target the one or more functions or activities mediated bythe effector domain to a particular target DNA sequence to which thenucleic acid binding domain specifically binds.

In some embodiments of the TALE polypeptides described herein, theactivity mediated by the effector domain is a biological activity. Forexample, in some embodiments the effector domain is a transcriptionalinhibitor (i.e., a repressor domain), such as an mSin interaction domain(SID). SID4X domain or a Kruppel-associated box (KRAB) or fragments ofthe KRAB domain. In some embodiments, the effector domain is an enhancerof transcription (i.e. an activation domain), such as the VP16, VP64 orp65 activation domain. In some embodiments, the nucleic acid binding islinked, for example, with an effector domain that includes but is notlimited to a transposase, integrase, recombinase, resolvase, invertase,protease, DNA methyltransferase, DNA demethylase, histone acetylase,histone deacetylase, nuclease, transcriptional repressor,transcriptional activator, transcription factor recruiting, proteinnuclear-localization signal or cellular uptake signal.

In some embodiments, the effector domain is a protein domain whichexhibits activities which include but are not limited to transposaseactivity, integrase activity, recombinase activity, resolvase activity,invertase activity, protease activity, DNA methyltransferase activity,DNA demethylase activity, histone acetylase activity, histonedeacetylase activity, nuclease activity, nuclear-localization signalingactivity, transcriptional repressor activity, transcriptional activatoractivity, transcription factor recruiting activity, or cellular uptakesignaling activity. Other preferred embodiments of the invention mayinclude any combination the activities described herein.

ZN-Finger Nucleases

Other preferred tools for genome editing for use in the context of thisinvention include zinc finger systems and TALE systems. One type ofprogrammable DNA-binding domain is provided by artificial zinc-finger(ZF) technology, which involves arrays of ZF modules to target newDNA-binding sites in the genome. Each finger module in a ZF arraytargets three DNA bases. A customized array of individual zinc fingerdomains is assembled into a ZF protein (ZFP).

ZFPs can comprise a functional domain. The first synthetic zinc fingernucleases (ZFNs) were developed by fusing a ZF protein to the catalyticdomain of the Type IIS restriction enzyme FokI. (Kim, Y. G. et al.,1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. U.S.A.91, 883-887; Kim, Y. G. et al., 1996, Hybrid restriction enzymes: zincfinger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. U.S.A.93, 1156-1160). Increased cleavage specificity can be attained withdecreased off target activity by use of paired ZFN heterodimers, eachtargeting different nucleotide sequences separated by a short spacer.(Doyon, Y. et al., 2011, Enhancing zinc-finger-nuclease activity withimproved obligate heterodimeric architectures. Nat. Methods 8, 74-79).ZFPs can also be designed as transcription activators and repressors andhave been used to target many genes in a wide variety of organisms.Exemplary methods of genome editing using ZFNs can be found for examplein U.S. Pat. Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978,6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719,7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626,all of which are specifically incorporated by reference.

Meganucleases

As disclosed herein editing can be made by way of meganucleases, whichare endodeoxyribonucleases characterized by a large recognition site(double-stranded DNA sequences of 12 to 40 base pairs). Exemplary methodfor using meganucleases can be found in U.S. Pat. Nos. 8,163,514;8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,369; and 8,129,134,which are specifically incorporated by reference.

Adoptive Cell Transfer

In certain embodiments, tumor cells are targeted by transferred immunecells. In certain embodiments, cancer stem cells are targeted. Incertain embodiments, adoptive cell transfer is performed in combinationwith a JAK/STAT inhibitor described herein. In certain embodiments,transferred cells are specific for a tumor antigen.

As used herein, “ACT”, “adoptive cell therapy” and “adoptive celltransfer” may be used interchangeably. Adoptive cell therapy (ACT) canrefer to the transfer of cells, most commonly immune-derived cells, backinto the same patient or into a new recipient host with the goal oftransferring the immunologic functionality and characteristics into thenew host. If possible, use of autologous cells helps the recipient byminimizing GVHD issues. The adoptive transfer of autologous tumorinfiltrating lymphocytes (TIL) (Besser et al., (2010) Clin. Cancer Res16 (9) 2646-55; Dudley et al., (2002) Science 298 (5594): 850-4; andDudley et al., (2005) Journal of Clinical Oncology 23 (10): 2346-57.) orgenetically re-directed peripheral blood mononuclear cells (Johnson etal., (2009) Blood 114 (3): 535-46; and Morgan et al., (2006) Science314(5796) 126-9) has been used to successfully treat patients withadvanced solid tumors, including melanoma and colorectal carcinoma, aswell as patients with CD19-expressing hematologic malignancies (Kalos etal., (2011) Science Translational Medicine 3 (95): 95ra73).

Aspects of the invention involve the adoptive transfer of immune systemcells, such as T cells, specific for selected antigens, such as tumorassociated antigens or tumor specific neoantigens (see Maus et al.,2014, Adoptive Immunotherapy for Cancer or Viruses, Annual Review ofImmunology, Vol. 32: 189-225; Rosenberg and Restifo, 2015, Adoptive celltransfer as personalized immunotherapy for human cancer, Science Vol.348 no. 6230 pp. 62-68; Restifo et al., 2015, Adoptive immunotherapy forcancer: harnessing the T cell response. Nat. Rev. Immunol. 12(4):269-281; and Jenson and Riddell, 2014, Design and implementation ofadoptive therapy with chimeric antigen receptor-modified T cells.Immunol Rev. 257(1): 127-144; and Rajasagi et al., 2014, Systematicidentification of personal tumor-specific neoantigens in chroniclymphocytic leukemia. Blood. 2014 Jul. 17; 124(3):453-62).

In certain embodiments, an antigen (such as a tumor antigen) to betargeted in adoptive cell therapy (such as particularly CAR or TCRT-cell therapy) of a disease (such as particularly of tumor or cancer)may be selected from a group consisting of: B cell maturation antigen(BCMA); PSA (prostate-specific antigen); prostate-specific membraneantigen (PSMA); PSCA (Prostate stem cell antigen); Tyrosine-proteinkinase transmembrane receptor ROR1; fibroblast activation protein (FAP);Tumor-associated glycoprotein 72 (TAG72); Carcinoembryonic antigen(CEA); Epithelial cell adhesion molecule (EPCAM); Mesothelin; HumanEpidermal growth factor Receptor 2 (ERBB2 (Her2/neu)); Prostate;Prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M);Insulin-like growth factor 1 receptor (IGF-1R); gplOO; BCR-ABL(breakpoint cluster region-Abelson); tyrosinase; New York esophagealsquamous cell carcinoma 1 (NY-ESO-1); κ-light chain, LAGE (L antigen);MAGE (melanoma antigen); Melanoma-associated antigen 1 (MAGE-A1); MAGEA3; MAGE A6; legumain; Human papillomavirus (HPV) E6; HPV E7; prostein;survivin; PCTA1 (Galectin 8); Melan-A/MART-1; Ras mutant; TRP-1(tyrosinase related protein 1, or gp75); Tyrosinase-related Protein 2(TRP2); TRP-2/INT2 (TRP-2/intron 2); RAGE (renal antigen); receptor foradvanced glycation end products 1 (RAGE1); Renal ubiquitous 1, 2 (RU1,RU2); intestinal carboxyl esterase (iCE); Heat shock protein 70-2(HSP70-2) mutant; thyroid stimulating hormone receptor (TSHR); CD123;CD171; CD19; CD20; CD22; CD26; CD30; CD33; CD44v7/8 (cluster ofdifferentiation 44, exons 7/8); CD53; CD92; CD100; CD148; CD150; CD200;CD261; CD262; CD362; CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319, and19A24); C-type lectin-like molecule-1 (CLL-1); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDG1cp(1-1)Cer); Tn antigen (Tn Ag);Fms-Like Tyrosine Kinase 3 (FLT3); CD38; CD138; CD44v6; B7H3 (CD276);KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2);Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen(PSCA); Protease Serine 21 (PRSS21); vascular endothelial growth factorreceptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growthfactor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4(SSEA-4); Mucin 1, cell surface associated (MUC1); mucin 16 (MUC16);epidermal growth factor receptor (EGFR); epidermal growth factorreceptor variant III (EGFRvIII); neural cell adhesion molecule (NCAM);carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit,Beta Type, 9 (LMP2); ephrin type-A receptor 2 (EphA2); Ephrin B2;Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3(aNeu5Ac(2-3)bDGalp(1-4)bDG1cp(1-1)Cer); TGS5; high molecularweight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside(OAcGD2); Folate receptor alpha; Folate receptor beta; tumor endothelialmarker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R);claudin 6 (CLDN6); G protein-coupled receptor class C group 5, member D(GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a;anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1(PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH);mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2);Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3(ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2(OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumorprotein (WT1); ETS translocation-variant gene 6, located on chromosome12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A(XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); CT(cancer/testis (antigen)); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53;p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin B1; Cyclin D1; v-myc avian myelocytomatosisviral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog FamilyMember C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor(Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma AntigenRecognized By T Cells-1 or 3 (SART1, SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint-1, -2, -3 or -4 (SSX1, SSX2, SSX3, SSX4);CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1(LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyteimmunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300molecule-like family member f (CD300LF); C-type lectin domain family 12member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-likemodule-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyteantigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); mousedouble minute 2 homolog (MDM2); livin; alphafetoprotein (AFP);transmembrane activator and CAML Interactor (TACI); B-cell activatingfactor receptor (BAFF-R); V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog (KRAS); immunoglobulin lambda-like polypeptide 1 (IGLL1); 707-AP(707 alanine proline); ART-4 (adenocarcinoma antigen recognized by T4cells); BAGE (B antigen; b-catenin/m, b-catenin/mutated); CAMEL(CTL-recognized antigen on melanoma); CAP1 (carcinoembryonic antigenpeptide 1); CASP-8 (caspase-8); CDC27m (cell-division cycle 27 mutated);CDK4/m (cycline-dependent kinase 4 mutated); Cyp-B (cyclophilin B); DAM(differentiation antigen melanoma); EGP-2 (epithelial glycoprotein 2);EGP-40 (epithelial glycoprotein 40); Erbb2, 3, 4 (erythroblasticleukemia viral oncogene homolog-2, -3, 4); FBP (folate binding protein);fAchR (Fetal acetylcholine receptor); G250 (glycoprotein 250); GAGE (Gantigen); GnT-V (N-acetylglucosaminyltransferase V); HAGE (helicoseantigen); ULA-A (human leukocyte antigen-A); HST2 (human signet ringtumor 2); KIAA0205; KDR (kinase insert domain receptor); LDLR/FUT (lowdensity lipid receptor/GDP L-fucose: b-D-galactosidase 2-a-Lfucosyltransferase); L1CAM (L1 cell adhesion molecule); MC1R(melanocortin 1 receptor); Myosin/m (myosin mutated); MUM-1, -2, -3(melanoma ubiquitous mutated 1, 2, 3); NA88-A (NA cDNA clone of patientM88); KG2D (Natural killer group 2, member D) ligands; oncofetal antigen(h5T4); p190 minor bcr-abl (protein of 190KD bcr-abl); Pml/RARa(promyelocytic leukaemia/retinoic acid receptor a); PRAME(preferentially expressed antigen of melanoma); SAGE (sarcoma antigen);TEL/AML1 (translocation Ets-family leukemia/acute myeloid leukemia 1);TPI/m (triosephosphate isomerase mutated); and any combination thereof.

In certain embodiments, an antigen to be targeted in adoptive celltherapy (such as particularly CAR or TCR T-cell therapy) of a disease(such as particularly of tumor or cancer) is a tumor-specific antigen(TSA).

In certain embodiments, an antigen to be targeted in adoptive celltherapy (such as particularly CAR or TCR T-cell therapy) of a disease(such as particularly of tumor or cancer) is a neoantigen.

In certain embodiments, an antigen to be targeted in adoptive celltherapy (such as particularly CAR or TCR T-cell therapy) of a disease(such as particularly of tumor or cancer) is a tumor-associated antigen(TAA).

In certain embodiments, an antigen to be targeted in adoptive celltherapy (such as particularly CAR or TCR T-cell therapy) of a disease(such as particularly of tumor or cancer) is a universal tumor antigen.In certain preferred embodiments, the universal tumor antigen isselected from the group consisting of: a human telomerase reversetranscriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2),cytochrome P450 1B 1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1),livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16(MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin(D1), and any combinations thereof.

In certain embodiments, an antigen (such as a tumor antigen) to betargeted in adoptive cell therapy (such as particularly CAR or TCRT-cell therapy) of a disease (such as particularly of tumor or cancer)may be selected from a group consisting of: CD19, BCMA, CLL-1, MAGE A3,MAGE A6, HPV E6, HPV E7, WT1, CD22, CD171, ROR1, MUC16, and SSX2. Incertain preferred embodiments, the antigen may be CD19. For example,CD19 may be targeted in hematologic malignancies, such as in lymphomas,more particularly in B-cell lymphomas, such as without limitation indiffuse large B-cell lymphoma, primary mediastinal b-cell lymphoma,transformed follicular lymphoma, marginal zone lymphoma, mantle celllymphoma, acute lymphoblastic leukemia including adult and pediatricALL, non-Hodgkin lymphoma, indolent non-Hodgkin lymphoma, or chroniclymphocytic leukemia. For example, BCMA may be targeted in multiplemyeloma or plasma cell leukemia. For example, CLL1 may be targeted inacute myeloid leukemia. For example, MAGE A3, MAGE A6, SSX2, and/or KRASmay be targeted in solid tumors. For example, HPV E6 and/or HPV E7 maybe targeted in cervical cancer or head and neck cancer. For example, WT1may be targeted in acute myeloid leukemia (AML), myelodysplasticsyndromes (MDS), chronic myeloid leukemia (CML), non-small cell lungcancer, breast, pancreatic, ovarian or colorectal cancers, ormesothelioma. For example, CD22 may be targeted in B cell malignancies,including non-Hodgkin lymphoma, diffuse large B-cell lymphoma, or acutelymphoblastic leukemia. For example, CD171 may be targeted inneuroblastoma, glioblastoma, or lung, pancreatic, or ovarian cancers.For example, ROR1 may be targeted in ROR1+ malignancies, includingnon-small cell lung cancer, triple negative breast cancer, pancreaticcancer, prostate cancer, ALL, chronic lymphocytic leukemia, or mantlecell lymphoma. For example, MUC16 may be targeted in MUC16ecto+epithelial ovarian, fallopian tube or primary peritoneal cancer.

Various strategies may for example be employed to genetically modify Tcells by altering the specificity of the T cell receptor (TCR) forexample by introducing new TCR α and β chains with selected peptidespecificity (see U.S. Pat. No. 8,697,854; PCT Patent Publications:WO2003020763, WO2004033685, WO2004044004, WO2005114215, WO2006000830,WO2008038002, WO2008039818, WO2004074322, WO2005113595, WO2006125962,WO2013166321, WO2013039889, WO2014018863, WO2014083173; U.S. Pat. No.8,088,379).

As an alternative to, or addition to, TCR modifications, chimericantigen receptors (CARs) may be used in order to generateimmunoresponsive cells, such as T cells, specific for selected targets,such as malignant cells, with a wide variety of receptor chimeraconstructs having been described (see U.S. Pat. Nos. 5,843,728;5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014;6,753,162; 8,211,422; and, PCT Publication WO9215322).

In general, CARs are comprised of an extracellular domain, atransmembrane domain, and an intracellular domain, wherein theextracellular domain comprises an antigen-binding domain that isspecific for a predetermined target. While the antigen-binding domain ofa CAR is often an antibody or antibody fragment (e.g., a single chainvariable fragment, scFv), the binding domain is not particularly limitedso long as it results in specific recognition of a target. For example,in some embodiments, the antigen-binding domain may comprise a receptor,such that the CAR is capable of binding to the ligand of the receptor.Alternatively, the antigen-binding domain may comprise a ligand, suchthat the CAR is capable of binding the endogenous receptor of thatligand.

The antigen-binding domain of a CAR is generally separated from thetransmembrane domain by a hinge or spacer. The spacer is also notparticularly limited, and it is designed to provide the CAR withflexibility. For example, a spacer domain may comprise a portion of ahuman Fc domain, including a portion of the CH3 domain, or the hingeregion of any immunoglobulin, such as IgA, IgD, IgE, IgG, or IgM, orvariants thereof. Furthermore, the hinge region may be modified so as toprevent off-target binding by FcRs or other potential interferingobjects. For example, the hinge may comprise an IgG4 Fc domain with orwithout a S228P, L235E, and/or N297Q mutation (according to Kabatnumbering) in order to decrease binding to FcRs. Additionalspacers/hinges include, but are not limited to, CD4, CD8, and CD28 hingeregions.

The transmembrane domain of a CAR may be derived either from a naturalor from a synthetic source. Where the source is natural, the domain maybe derived from any membrane bound or transmembrane protein.Transmembrane regions of particular use in this disclosure may bederived from CD8, CD28, CD3, CD45, CD4, CD5, CDS, CD9, CD 16, CD22,CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, TCR. Alternatively,the transmembrane domain may be synthetic, in which case it willcomprise predominantly hydrophobic residues such as leucine and valine.Preferably a triplet of phenylalanine, tryptophan and valine will befound at each end of a synthetic transmembrane domain. Optionally, ashort oligo- or polypeptide linker, preferably between 2 and 10 aminoacids in length may form the linkage between the transmembrane domainand the cytoplasmic signaling domain of the CAR. A glycine-serinedoublet provides a particularly suitable linker.

Alternative CAR constructs may be characterized as belonging tosuccessive generations. First-generation CARs typically consist of asingle-chain variable fragment of an antibody specific for an antigen,for example comprising a VL linked to a VH of a specific antibody,linked by a flexible linker, for example by a CD8a hinge domain and aCD8a transmembrane domain, to the transmembrane and intracellularsignaling domains of either CD3ζ or FcRγ (scFv-CD3ζ or scFv-FcRγ; seeU.S. Pat. Nos. 7,741,465; 5,912,172; 5,906,936). Second-generation CARsincorporate the intracellular domains of one or more costimulatorymolecules, such as CD28, OX40 (CD134), or 4-1BB (CD137) within theendodomain (for example scFv-CD28/OX40/4-1BB-CD3; see U.S. Pat. Nos.8,911,993; 8,916,381; 8,975,071; 9,101,584; 9,102,760; 9,102,761).Third-generation CARs include a combination of costimulatoryendodomains, such a CD3-chain, CD97, GDI la-CD18, CD2, ICOS, CD27,CD154, CDS, OX40, 4-1BB, CD2, CD7, LIGHT, LFA-1, NKG2C, B7-H3, CD30,CD40, PD-1, or CD28 signaling domains (for example scFv-CD28-4-1BB-CD3ζor scFv-CD28-OX40-CD3; see U.S. Pat. Nos. 8,906,682; 8,399,645;5,686,281; PCT Publication No. WO2014134165; PCT Publication No.WO2012079000). In certain embodiments, the primary signaling domaincomprises a functional signaling domain of a protein selected from thegroup consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, commonFcR gamma (FCERIG), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fc gammaRIIa, DAP10, and DAP12. In certain preferred embodiments, the primarysignaling domain comprises a functional signaling domain of CD3ζ orFcRγ. In certain embodiments, the one or more costimulatory signalingdomains comprise a functional signaling domain of a protein selected,each independently, from the group consisting of: CD27, CD28, 4-1BB(CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand thatspecifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR),SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta,IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM,CD11b, ITGAX, CD11 c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2,TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile),CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69,SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46,and NKG2D. In certain embodiments, the one or more costimulatorysignaling domains comprise a functional signaling domain of a proteinselected, each independently, from the group consisting of: 4-1BB, CD27,and CD28. In certain embodiments, a chimeric antigen receptor may havethe design as described in U.S. Pat. No. 7,446,190, comprising anintracellular domain of CD3ζ chain (such as amino acid residues 52-163of the human CD3 zeta chain, as shown in SEQ ID NO: 14 of U.S. Pat. No.7,446,190), a signaling region from CD28 and an antigen-binding element(or portion or domain; such as scFv). The CD28 portion, when between thezeta chain portion and the antigen-binding element, may suitably includethe transmembrane and signaling domains of CD28 (such as amino acidresidues 114-220 of SEQ ID NO: 10, full sequence shown in SEQ ID NO: 6of U.S. Pat. No. 7,446,190; these can include the following portion ofCD28 as set forth in Genbank identifier NM_006139 (sequence version 1, 2or 3):

IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP PRDFAAYRS)).Alternatively, when the zeta sequence lies between the CD28 sequence andthe antigen-binding element, intracellular domain of CD28 can be usedalone (such as amino sequence set forth in SEQ ID NO: 9 of U.S. Pat. No.7,446,190). Hence, certain embodiments employ a CAR comprising (a) azeta chain portion comprising the intracellular domain of human CD3chain, (b) a costimulatory signaling region, and (c) an antigen-bindingelement (or portion or domain), wherein the costimulatory signalingregion comprises the amino acid sequence encoded by SEQ ID NO: 6 of U.S.Pat. No. 7,446,190.

Alternatively, costimulation may be orchestrated by expressing CARs inantigen-specific T cells, chosen so as to be activated and expandedfollowing engagement of their native αβTCR, for example by antigen onprofessional antigen-presenting cells, with attendant costimulation. Inaddition, additional engineered receptors may be provided on theimmunoresponsive cells, for example to improve targeting of a T-cellattack and/or minimize side effects

By means of an example and without limitation, Kochenderfer et al.,(2009) J Immunother. 32 (7): 689-702 described anti-CD19 chimericantigen receptors (CAR). FMC63-28Z CAR contained a single chain variableregion moiety (scFv) recognizing CD19 derived from the FMC63 mousehybridoma (described in Nicholson et al., (1997) Molecular Immunology34: 1157-1165), a portion of the human CD28 molecule, and theintracellular component of the human TCR-molecule. FMC63-CD828BBZ CARcontained the FMC63 scFv, the hinge and transmembrane regions of the CD8molecule, the cytoplasmic portions of CD28 and 4-1BB, and thecytoplasmic component of the TCR-molecule. The exact sequence of theCD28 molecule included in the FMC63-28Z CAR corresponded to Genbankidentifier NM_006139; the sequence included all amino acids startingwith the amino acid sequence IEVMYPPPY (SEQ ID No: 7) and continuing allthe way to the carboxy-terminus of the protein. To encode the anti-CD19scFv component of the vector, the authors designed a DNA sequence whichwas based on a portion of a previously published CAR (Cooper et al.,(2003) Blood 101: 1637-1644). This sequence encoded the followingcomponents in frame from the 5′ end to the 3′ end: an XhoI site, thehuman granulocyte-macrophage colony-stimulating factor (GM-CSF) receptorα-chain signal sequence, the FMC63 light chain variable region (as inNicholson et al., supra), a linker peptide (as in Cooper et al., supra),the FMC63 heavy chain variable region (as in Nicholson et al., supra),and a NotI site. A plasmid encoding this sequence was digested with XhoIand NotI. To form the MSGV-FMC63-28Z retroviral vector, the XhoI andNotI-digested fragment encoding the FMC63 scFv was ligated into a secondXhoI and NotI-digested fragment that encoded the MSGV retroviralbackbone (as in Hughes et al., (2005) Human Gene Therapy 16: 457-472) aswell as part of the extracellular portion of human CD28, the entiretransmembrane and cytoplasmic portion of human CD28, and the cytoplasmicportion of the human TCR-molecule (as in Maher et al., 2002) NatureBiotechnology 20: 70-75). The FMC63-28Z CAR is included in the KTE-C19(axicabtagene ciloleucel) anti-CD19 CAR-T therapy product in developmentby Kite Pharma, Inc. for the treatment of inter alia patients withrelapsed/refractory aggressive B-cell non-Hodgkin lymphoma (NHL).Accordingly, in certain embodiments, cells intended for adoptive celltherapies, more particularly immunoresponsive cells such as T cells, mayexpress the FMC63-28Z CAR as described by Kochenderfer et al. (supra).Hence, in certain embodiments, cells intended for adoptive celltherapies, more particularly immunoresponsive cells such as T cells, maycomprise a CAR comprising an extracellular antigen-binding element (orportion or domain; such as scFv) that specifically binds to an antigen,an intracellular signaling domain comprising an intracellular domain ofa CD3 chain, and a costimulatory signaling region comprising a signalingdomain of CD28. Preferably, the CD28 amino acid sequence is as set forthin Genbank identifier NM_006139 (sequence version 1,2 or 3) startingwith the amino acid sequence IEVMYPPPY and continuing all the way to thecarboxy-terminus of the protein. The sequence is reproduced herein:IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID No: 8).Preferably, the antigen is CD19, more preferably the antigen-bindingelement is an anti-CD19 scFv, even more preferably the anti-CD19 scFv asdescribed by Kochenderfer et al. (supra).

Additional anti-CD19 CARs are further described in WO2015187528. Moreparticularly Example 1 and Table 1 of WO2015187528, incorporated byreference herein, demonstrate the generation of anti-CD19 CARs based ona fully human anti-CD19 monoclonal antibody (47G4, as described inUS20100104509) and murine anti-CD19 monoclonal antibody (as described inNicholson et al. and explained above). Various combinations of a signalsequence (human CD8-alpha or GM-CSF receptor), extracellular andtransmembrane regions (human CD8-alpha) and intracellular T-cellsignalling domains (CD28-CD3ζ; 4-1BB-CD3ζ; CD27-CD3ζ; CD28-CD27-CD3ζ,4-1BB-CD27-CD3ζ; CD27-4-1BB-CD3ζ; CD28-CD27-FccRI gamma chain; orCD28-FccRI gamma chain) were disclosed. Hence, in certain embodiments,cells intended for adoptive cell therapies, more particularlyimmunoresponsive cells such as T cells, may comprise a CAR comprising anextracellular antigen-binding element that specifically binds to anantigen, an extracellular and transmembrane region as set forth in Table1 of WO2015187528 and an intracellular T-cell signalling domain as setforth in Table 1 of WO2015187528. Preferably, the antigen is CD19, morepreferably the antigen-binding element is an anti-CD19 scFv, even morepreferably the mouse or human anti-CD19 scFv as described in Example 1of WO2015187528. In certain embodiments, the CAR comprises, consistsessentially of or consists of an amino acid sequence of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, or SEQ ID NO: 13 as set forth in Table 1 of WO2015187528.

In certain embodiments, the immune cell may, in addition to a CAR orexogenous TCR as described herein, further comprise a chimericinhibitory receptor (inhibitory CAR) that specifically binds to a secondtarget antigen and is capable of inducing an inhibitory orimmunosuppressive or repressive signal to the cell upon recognition ofthe second target antigen. In certain embodiments, the chimericinhibitory receptor comprises an extracellular antigen-binding element(or portion or domain) configured to specifically bind to a targetantigen, a transmembrane domain, and an intracellular immunosuppressiveor repressive signaling domain. In certain embodiments, the secondtarget antigen is an antigen that is not expressed on the surface of acancer cell or infected cell or the expression of which is downregulatedon a cancer cell or an infected cell. In certain embodiments, the secondtarget antigen is an MHC-class I molecule. In certain embodiments, theintracellular signaling domain comprises a functional signaling portionof an immune checkpoint molecule, such as for example PD-1 or CTLA4.Advantageously, the inclusion of such inhibitory CAR reduces the chanceof the engineered immune cells attacking non-target (e.g., non-cancer)tissues.

Alternatively, T-cells expressing CARs may be further modified to reduceor eliminate expression of endogenous TCRs in order to reduce off-targeteffects. Reduction or elimination of endogenous TCRs can reduceoff-target effects and increase the effectiveness of the T cells (U.S.Pat. No. 9,181,527). T cells stably lacking expression of a functionalTCR may be produced using a variety of approaches. T cells internalize,sort, and degrade the entire T cell receptor as a complex, with ahalf-life of about 10 hours in resting T cells and 3 hours in stimulatedT cells (von Essen, M. et al. 2004. J. Immunol. 173:384-393). Properfunctioning of the TCR complex requires the proper stoichiometric ratioof the proteins that compose the TCR complex. TCR function also requirestwo functioning TCR zeta proteins with ITAM motifs. The activation ofthe TCR upon engagement of its WIC-peptide ligand requires theengagement of several TCRs on the same T cell, which all must signalproperly. Thus, if a TCR complex isdestabilized with proteins that donot associate properly or cannot signal optimally, the T cell will notbecome activated sufficiently to begin a cellular response.

Accordingly, in some embodiments, TCR expression may eliminated usingRNA interference (e.g., shRNA, siRNA, miRNA, etc.), CRISPR, or othermethods that target the nucleic acids encoding specific TCRs (e.g.,TCR-α and TCR-β) and/or CD3 chains in primary T cells. By blockingexpression of one or more of these proteins, the T cell will no longerproduce one or more of the key components of the TCR complex, therebydestabilizing the TCR complex and preventing cell surface expression ofa functional TCR.

In some instances, CAR may also comprise a switch mechanism forcontrolling expression and/or activation of the CAR. For example, a CARmay comprise an extracellular, transmembrane, and intracellular domain,in which the extracellular domain comprises a target-specific bindingelement that comprises a label, binding domain, or tag that is specificfor a molecule other than the target antigen that is expressed on or bya target cell. In such embodiments, the specificity of the CAR isprovided by a second construct that comprises a target antigen bindingdomain (e.g., an scFv or a bispecific antibody that is specific for boththe target antigen and the label or tag on the CAR) and a domain that isrecognized by or binds to the label, binding domain, or tag on the CAR.See, e.g., WO 2013/044225, WO 2016/000304, WO 2015/057834, WO2015/057852, WO 2016/070061, U.S. Pat. No. 9,233,125, US 2016/0129109.In this way, a T-cell that expresses the CAR can be administered to asubject, but the CAR cannot bind its target antigen until the secondcomposition comprising an antigen-specific binding domain isadministered.

Alternative switch mechanisms include CARs that require multimerizationin order to activate their signaling function (see, e.g., US2015/0368342, US 2016/0175359, US 2015/0368360) and/or an exogenoussignal, such as a small molecule drug (US 2016/0166613, Yung et al.,Science, 2015), in order to elicit a T-cell response. Some CARs may alsocomprise a “suicide switch” to induce cell death of the CAR T-cellsfollowing treatment (Buddee et al., PLoS One, 2013) or to downregulateexpression of the CAR following binding to the target antigen (WO2016/011210).

Alternative techniques may be used to transform target immunoresponsivecells, such as protoplast fusion, lipofection, transfection orelectroporation. A wide variety of vectors may be used, such asretroviral vectors, lentiviral vectors, adenoviral vectors,adeno-associated viral vectors, plasmids or transposons, such as aSleeping Beauty transposon (see U.S. Pat. Nos. 6,489,458; 7,148,203;7,160,682; 7,985,739; 8,227,432), may be used to introduce CARs, forexample using 2nd generation antigen-specific CARs signaling through CD3and either CD28 or CD137. Viral vectors may for example include vectorsbased on HIV, SV40, EBV, HSV or BPV.

Cells that are targeted for transformation may for example include Tcells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL),regulatory T cells, human embryonic stem cells, tumor-infiltratinglymphocytes (TIL) or a pluripotent stem cell from which lymphoid cellsmay be differentiated. T cells expressing a desired CAR may for examplebe selected through co-culture with γ-irradiated activating andpropagating cells (AaPC), which co-express the cancer antigen andco-stimulatory molecules. The engineered CAR T-cells may be expanded,for example by co-culture on AaPC in presence of soluble factors, suchas IL-2 and IL-21. This expansion may for example be carried out so asto provide memory CAR+ T cells (which may for example be assayed bynon-enzymatic digital array and/or multi-panel flow cytometry). In thisway, CAR T cells may be provided that have specific cytotoxic activityagainst antigen-bearing tumors (optionally in conjunction withproduction of desired chemokines such as interferon-γ). CAR T cells ofthis kind may for example be used in animal models, for example to treattumor xenografts.

In certain embodiments, ACT includes co-transferring CD4+Th1 cells andCD8+ CTLs to induce a synergistic antitumour response (see, e.g., Li etal., Adoptive cell therapy with CD4+T helper 1 cells and CD8+ cytotoxicT cells enhances complete rejection of an established tumour, leading togeneration of endogenous memory responses to non-targeted tumourepitopes. Clin Transl Immunology. 2017 October; 6(10): e160).

In certain embodiments, Th17 cells are transferred to a subject in needthereof. Th17 cells have been reported to directly eradicate melanomatumors in mice to a greater extent than Th1 cells (Muranski P, et al.,Tumor-specific Th17-polarized cells eradicate large establishedmelanoma. Blood. 2008 Jul. 15; 112(2):362-73; and Martin-Orozco N, etal., T helper 17 cells promote cytotoxic T cell activation in tumorimmunity. Immunity. 2009 Nov. 20; 31(5):787-98). Those studies involvedan adoptive T cell transfer (ACT) therapy approach, which takesadvantage of CD4⁺ T cells that express a TCR recognizing tyrosinasetumor antigen. Exploitation of the TCR leads to rapid expansion of Th17populations to large numbers ex vivo for reinfusion into the autologoustumor-bearing hosts.

In certain embodiments, ACT may include autologous iPSC-based vaccines,such as irradiated iPSCs in autologous anti-tumor vaccines (see e.g.,Kooreman, Nigel G. et al., Autologous iPSC-Based Vaccines ElicitAnti-tumor Responses In Vivo, Cell Stem Cell 22, 1-13,2018,doi.org/10.1016/j.stem.2018.01.016).

Unlike T-cell receptors (TCRs) that are MHC restricted, CARs canpotentially bind any cell surface-expressed antigen and can thus be moreuniversally used to treat patients (see Irving et al., EngineeringChimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don'tForget the Fuel, Front. Immunol., 3 Apr. 2017,doi.org/10.3389/fimmu.2017.00267). In certain embodiments, in theabsence of endogenous T-cell infiltrate (e.g., due to aberrant antigenprocessing and presentation), which precludes the use of TIL therapy andimmune checkpoint blockade, the transfer of CAR T-cells may be used totreat patients (see, e.g., Hinrichs C S, Rosenberg S A. Exploiting thecurative potential of adoptive T-cell therapy for cancer. Immunol Rev(2014) 257(1):56-71. doi:10.1111/imr.12132).

Approaches such as the foregoing may be adapted to provide methods oftreating and/or increasing survival of a subject having a disease, suchas a neoplasia, for example by administering an effective amount of animmunoresponsive cell comprising an antigen recognizing receptor thatbinds a selected antigen, wherein the binding activates theimmunoresponsive cell, thereby treating or preventing the disease (suchas a neoplasia, a pathogen infection, an autoimmune disorder, or anallogeneic transplant reaction).

In certain embodiments, the treatment can be administered afterlymphodepleting pretreatment in the form of chemotherapy (typically acombination of cyclophosphamide and fludarabine) or radiation therapy.Initial studies in ACT had short lived responses and the transferredcells did not persist in vivo for very long (Houot et al., T-cell-basedimmunotherapy: adoptive cell transfer and checkpoint inhibition. CancerImmunol Res (2015) 3(10):1115-22; and Kamta et al., Advancing CancerTherapy with Present and Emerging Immuno-Oncology Approaches. Front.Oncol. (2017) 7:64). Immune suppressor cells like Tregs and MDSCs mayattenuate the activity of transferred cells by outcompeting them for thenecessary cytokines. Not being bound by a theory lymphodepletingpretreatment may eliminate the suppressor cells allowing the TILs topersist.

In one embodiment, the treatment can be administrated into patientsundergoing an immunosuppressive treatment. The cells or population ofcells, may be made resistant to at least one immunosuppressive agent dueto the inactivation of a gene encoding a receptor for suchimmunosuppressive agent. Not being bound by a theory, theimmunosuppressive treatment should help the selection and expansion ofthe immunoresponsive or T cells according to the invention within thepatient.

In certain embodiments, the treatment can be administered before primarytreatment (e.g., surgery or radiation therapy) to shrink a tumor beforethe primary treatment. In another embodiment, the treatment can beadministered after primary treatment to remove any remaining cancercells.

In certain embodiments, immunometabolic barriers can be targetedtherapeutically prior to and/or during ACT to enhance responses to ACTor CAR T-cell therapy and to support endogenous immunity (see, e.g.,Irving et al., Engineering Chimeric Antigen Receptor T-Cells for Racingin Solid Tumors: Don't Forget the Fuel, Front. Immunol., 3 Apr. 2017,doi.org/10.3389/fimmu.2017.00267).

The administration of cells or population of cells, such as immunesystem cells or cell populations, such as more particularlyimmunoresponsive cells or cell populations, as disclosed herein may becarried out in any convenient manner, including by aerosol inhalation,injection, ingestion, transfusion, implantation or transplantation. Thecells or population of cells may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, intrathecally, by intravenous orintralymphatic injection, or intraperitoneally. In some embodiments, thedisclosed CARs may be delivered or administered into a cavity formed bythe resection of tumor tissue (i.e. intracavity delivery) or directlyinto a tumor prior to resection (i.e. intratumoral delivery). In oneembodiment, the cell compositions of the present invention arepreferably administered by intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of 10⁴-10⁹ cells per kg body weight, preferably 10⁵to 10⁶ cells/kg body weight including all integer values of cell numberswithin those ranges. Dosing in CAR T cell therapies may for exampleinvolve administration of from 10⁶ to 10⁹ cells/kg, with or without acourse of lymphodepletion, for example with cyclophosphamide. The cellsor population of cells can be administrated in one or more doses. Inanother embodiment, the effective amount of cells are administrated as asingle dose. In another embodiment, the effective amount of cells areadministrated as more than one dose over a period time. Timing ofadministration is within the judgment of managing physician and dependson the clinical condition of the patient. The cells or population ofcells may be obtained from any source, such as a blood bank or a donor.While individual needs vary, determination of optimal ranges ofeffective amounts of a given cell type for a particular disease orconditions are within the skill of one in the art. An effective amountmeans an amount which provides a therapeutic or prophylactic benefit.The dosage administrated will be dependent upon the age, health andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment and the nature of the effect desired.

In another embodiment, the effective amount of cells or compositioncomprising those cells are administrated parenterally. Theadministration can be an intravenous administration. The administrationcan be directly done by injection within a tumor.

To guard against possible adverse reactions, engineered immunoresponsivecells may be equipped with a transgenic safety switch, in the form of atransgene that renders the cells vulnerable to exposure to a specificsignal. For example, the herpes simplex viral thymidine kinase (TK) genemay be used in this way, for example by introduction into allogeneic Tlymphocytes used as donor lymphocyte infusions following stem celltransplantation (Greco, et al., Improving the safety of cell therapywith the TK-suicide gene. Front. Pharmacol. 2015; 6: 95). In such cells,administration of a nucleoside prodrug such as ganciclovir or acyclovircauses cell death. Alternative safety switch constructs includeinducible caspase 9, for example triggered by administration of asmall-molecule dimerizer that brings together two nonfunctional icasp9molecules to form the active enzyme. A wide variety of alternativeapproaches to implementing cellular proliferation controls have beendescribed (see U.S. Patent Publication No. 20130071414; PCT PatentPublication WO2011146862; PCT Patent Publication WO2014011987; PCTPatent Publication WO2013040371; Zhou et al. BLOOD, 2014,123/25:3895-3905; Di Stasi et al., The New England Journal of Medicine2011; 365:1673-1683; Sadelain M, The New England Journal of Medicine2011; 365:1735-173; Ramos et al., Stem Cells 28(6):1107-15 (2010)).

In a further refinement of adoptive therapies, genome editing may beused to tailor immunoresponsive cells to alternative implementations,for example providing edited CAR T cells (see Poirot et al., 2015,Multiplex genome edited T-cell manufacturing platform for“off-the-shelf” adoptive T-cell immunotherapies, Cancer Res 75 (18):3853; Ren et al., 2016, Multiplex genome editing to generate universalCAR T cells resistant to PD1 inhibition, Clin Cancer Res. 2016 Nov. 4;and Qasim et al., 2017, Molecular remission of infant B-ALL afterinfusion of universal TALEN gene-edited CAR T cells, Sci Transl Med.2017 Jan. 25; 9(374)). Cells may be edited using any CRISPR system andmethod of use thereof as described herein. CRISPR systems may bedelivered to an immune cell by any method described herein. In preferredembodiments, cells are edited ex vivo and transferred to a subject inneed thereof. Immunoresponsive cells, CAR T cells or any cells used foradoptive cell transfer may be edited. Editing may be performed forexample to insert or knock-in an exogenous gene, such as an exogenousgene encoding a CAR or a TCR, at a preselected locus in a cell; toeliminate potential alloreactive T-cell receptors (TCR) or to preventinappropriate pairing between endogenous and exogenous TCR chains, suchas to knock-out or knock-down expression of an endogenous TCR in a cell;to disrupt the target of a chemotherapeutic agent in a cell; to block animmune checkpoint, such as to knock-out or knock-down expression of animmune checkpoint protein or receptor in a cell; to knock-out orknock-down expression of other gene or genes in a cell, the reducedexpression or lack of expression of which can enhance the efficacy ofadoptive therapies using the cell; to knock-out or knock-down expressionof an endogenous gene in a cell, said endogenous gene encoding anantigen targeted by an exogenous CAR or TCR; to knock-out or knock-downexpression of one or more MHC constituent proteins in a cell; toactivate a T cell; to modulate cells such that the cells are resistantto exhaustion or dysfunction; and/or increase the differentiation and/orproliferation of functionally exhausted or dysfunctional CD8+ T-cells(see PCT Patent Publications: WO2013176915, WO2014059173, WO2014172606,WO2014184744, and WO2014191128). Editing may result in inactivation of agene.

By inactivating a gene it is intended that the gene of interest is notexpressed in a functional protein form. In a particular embodiment, theCRISPR system specifically catalyzes cleavage in one targeted genethereby inactivating said targeted gene. The nucleic acid strand breakscaused are commonly repaired through the distinct mechanisms ofhomologous recombination or non-homologous end joining (NHEJ). However,NHEJ is an imperfect repair process that often results in changes to theDNA sequence at the site of the cleavage. Repair via non-homologous endjoining (NHEJ) often results in small insertions or deletions (Indel)and can be used for the creation of specific gene knockouts. Cells inwhich a cleavage induced mutagenesis event has occurred can beidentified and/or selected by well-known methods in the art.

Hence, in certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toinsert or knock-in an exogenous gene, such as an exogenous gene encodinga CAR or a TCR, at a preselected locus in a cell. Conventionally,nucleic acid molecules encoding CARs or TCRs are transfected ortransduced to cells using randomly integrating vectors, which, dependingon the site of integration, may lead to clonal expansion, oncogenictransformation, variegated transgene expression and/or transcriptionalsilencing of the transgene. Directing of transgene(s) to a specificlocus in a cell can minimize or avoid such risks and advantageouslyprovide for uniform expression of the transgene(s) by the cells. Withoutlimitation, suitable ‘safe harbor’ loci for directed transgeneintegration include CCR5 or AAVS1. Homology-directed repair (HDR)strategies are known and described elsewhere in this specificationallowing to insert transgenes into desired loci.

Further suitable loci for insertion of transgenes, in particular CAR orexogenous TCR transgenes, include without limitation loci comprisinggenes coding for constituents of endogenous T-cell receptor, such asT-cell receptor alpha locus (TRA) or T-cell receptor beta locus (TRB),for example T-cell receptor alpha constant (TRAC) locus, T-cell receptorbeta constant 1 (TRBC1) locus or T-cell receptor beta constant 2 (TRBC1)locus.

Advantageously, insertion of a transgene into such locus cansimultaneously achieve expression of the transgene, potentiallycontrolled by the endogenous promoter, and knock-out expression of theendogenous TCR. This approach has been exemplified in Eyquem et al.,(2017) Nature 543: 113-117, wherein the authors used CRISPR/Cas9 geneediting to knock-in a DNA molecule encoding a CD19-specific CAR into theTRAC locus downstream of the endogenous promoter; the CAR-T cellsobtained by CRISPR were significantly superior in terms of reduced tonicCAR signaling and exhaustion.

T cell receptors (TCR) are cell surface receptors that participate inthe activation of T cells in response to the presentation of antigen.The TCR is generally made from two chains, α and β, which assemble toform a heterodimer and associates with the CD3-transducing subunits toform the T cell receptor complex present on the cell surface. Each α andβ chain of the TCR consists of an immunoglobulin-like N-terminalvariable (V) and constant (C) region, a hydrophobic transmembranedomain, and a short cytoplasmic region. As for immunoglobulin molecules,the variable region of the α and β chains are generated by V(D)Jrecombination, creating a large diversity of antigen specificitieswithin the population of T cells. However, in contrast toimmunoglobulins that recognize intact antigen, T cells are activated byprocessed peptide fragments in association with an MHC molecule,introducing an extra dimension to antigen recognition by T cells, knownas MHC restriction. Recognition of MHC disparities between the donor andrecipient through the T cell receptor leads to T cell proliferation andthe potential development of graft versus host disease (GVHD). Theinactivation of TCRα or TCRβ can result in the elimination of the TCRfrom the surface of T cells preventing recognition of alloantigen andthus GVHD. However, TCR disruption generally results in the eliminationof the CD3 signaling component and alters the means of further T cellexpansion.

Hence, in certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toknock-out or knock-down expression of an endogenous TCR in a cell. Forexample, NHEJ-based or HDR-based gene editing approaches can be employedto disrupt the endogenous TCR alpha and/or beta chain genes. Forexample, gene editing system or systems, such as CRISPR/Cas system orsystems, can be designed to target a sequence found within the TCR betachain conserved between the beta 1 and beta 2 constant region genes(TRBC1 and TRBC2) and/or to target the constant region of the TCR alphachain (TRAC) gene.

Allogeneic cells are rapidly rejected by the host immune system. It hasbeen demonstrated that, allogeneic leukocytes present in non-irradiatedblood products will persist for no more than 5 to 6 days (Boni, Muranskiet al. 2008 Blood 1; 112(12):4746-54). Thus, to prevent rejection ofallogeneic cells, the host's immune system usually has to be suppressedto some extent. However, in the case of adoptive cell transfer the useof immunosuppressive drugs also have a detrimental effect on theintroduced therapeutic T cells. Therefore, to effectively use anadoptive immunotherapy approach in these conditions, the introducedcells would need to be resistant to the immunosuppressive treatment.Thus, in a particular embodiment, the present invention furthercomprises a step of modifying T cells to make them resistant to animmunosuppressive agent, preferably by inactivating at least one geneencoding a target for an immunosuppressive agent. An immunosuppressiveagent is an agent that suppresses immune function by one of severalmechanisms of action. An immunosuppressive agent can be, but is notlimited to a calcineurin inhibitor, a target of rapamycin, aninterleukin-2 receptor α-chain blocker, an inhibitor of inosinemonophosphate dehydrogenase, an inhibitor of dihydrofolic acidreductase, a corticosteroid or an immunosuppressive antimetabolite. Thepresent invention allows conferring immunosuppressive resistance to Tcells for immunotherapy by inactivating the target of theimmunosuppressive agent in T cells. As non-limiting examples, targetsfor an immunosuppressive agent can be a receptor for animmunosuppressive agent such as: CD52, glucocorticoid receptor (GR), aFKBP family gene member and a cyclophilin family gene member.

In certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toblock an immune checkpoint, such as to knock-out or knock-downexpression of an immune checkpoint protein or receptor in a cell. Immunecheckpoints are inhibitory pathways that slow down or stop immunereactions and prevent excessive tissue damage from uncontrolled activityof immune cells. In certain embodiments, the immune checkpoint targetedis the programmed death-1 (PD-1 or CD279) gene (PDCD1). In otherembodiments, the immune checkpoint targeted is cytotoxicT-lymphocyte-associated antigen (CTLA-4). In additional embodiments, theimmune checkpoint targeted is another member of the CD28 and CTLA4 Igsuperfamily such as BTLA, LAG3, ICOS, PDL1 or KIR. In further additionalembodiments, the immune checkpoint targeted is a member of the TNFRsuperfamily such as CD40, OX40, CD137, GITR, CD27 or TIM-3.

Additional immune checkpoints include Src homology 2 domain-containingprotein tyrosine phosphatase 1 (SHP-1) (Watson H A, et al., SHP-1: thenext checkpoint target for cancer immunotherapy? Biochem Soc Trans. 2016Apr. 15; 44(2):356-62). SHP-1 is a widely expressed inhibitory proteintyrosine phosphatase (PTP). In T-cells, it is a negative regulator ofantigen-dependent activation and proliferation. It is a cytosolicprotein, and therefore not amenable to antibody-mediated therapies, butits role in activation and proliferation makes it an attractive targetfor genetic manipulation in adoptive transfer strategies, such aschimeric antigen receptor (CAR) T cells. Immune checkpoints may alsoinclude T cell immunoreceptor with Ig and ITIM domains(TIGIT/Vstm3/WUCAM/VSIG9) and VISTA (Le Mercier I, et al., (2015) BeyondCTLA-4 and PD-1, the generation Z of negative checkpoint regulators.Front. Immunol. 6:418).

WO2014172606 relates to the use of MT1 and/or MT2 inhibitors to increaseproliferation and/or activity of exhausted CD8+ T-cells and to decreaseCD8+ T-cell exhaustion (e.g., decrease functionally exhausted orunresponsive CD8+ immune cells). In certain embodiments,metallothioneins are targeted by gene editing in adoptively transferredT cells.

In certain embodiments, targets of gene editing may be at least onetargeted locus involved in the expression of an immune checkpointprotein. Such targets may include, but are not limited to CTLA4, PPP2CA,PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2,BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244 (2B4),TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS,TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, bTGIF1,IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3,PRDM1, BATF, VISTA, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, MT1, MT2, CD40,OX40, CD137, GITR, CD27, SHP-1, TIM-3, CEACAM-1, CEACAM-3, or CEACAM-5.In preferred embodiments, the gene locus involved in the expression ofPD-1 or CTLA-4 genes is targeted. In other preferred embodiments,combinations of genes are targeted, such as but not limited to PD-1 andTIGIT.

By means of an example and without limitation, WO2016196388 concerns anengineered T cell comprising (a) a genetically engineered antigenreceptor that specifically binds to an antigen, which receptor may be aCAR; and (b) a disrupted gene encoding a PD-L1, an agent for disruptionof a gene encoding a PD-L1, and/or disruption of a gene encoding PD-L1,wherein the disruption of the gene may be mediated by a gene editingnuclease, a zinc finger nuclease (ZFN), CRISPR/Cas9 and/or TALEN.WO2015142675 relates to immune effector cells comprising a CAR incombination with an agent (such as CRISPR, TALEN or ZFN) that increasesthe efficacy of the immune effector cells in the treatment of cancer,wherein the agent may inhibit an immune inhibitory molecule, such asPD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4,TGFR beta, CEACAM-1, CEACAM-3, or CEACAM-5. Ren et al., (2017) ClinCancer Res 23 (9) 2255-2266 performed lentiviral delivery of CAR andelectro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, β-2microglobulin (B2M) and PD1 simultaneously, to generate gene-disruptedallogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.

In certain embodiments, cells may be engineered to express a CAR,wherein expression and/or function of methylcytosine dioxygenase genes(TET1, TET2 and/or TET3) in the cells has been reduced or eliminated,such as by CRISPR, ZNF or TALEN (for example, as described inWO201704916).

In certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toknock-out or knock-down expression of an endogenous gene in a cell, saidendogenous gene encoding an antigen targeted by an exogenous CAR or TCR,thereby reducing the likelihood of targeting of the engineered cells. Incertain embodiments, the targeted antigen may be one or more antigenselected from the group consisting of CD38, CD138, CS-1, CD33, CD26,CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, CD362, humantelomerase reverse transcriptase (hTERT), survivin, mouse double minute2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumorgene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen(CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen(PSMA), p53, cyclin (D1), B cell maturation antigen (BCMA),transmembrane activator and CAML Interactor (TACI), and B-cellactivating factor receptor (BAFF-R) (for example, as described inWO2016011210 and WO2017011804).

In certain embodiments, editing of cells (such as by CRISPR/Cas),particularly cells intended for adoptive cell therapies, moreparticularly immunoresponsive cells such as T cells, may be performed toknock-out or knock-down expression of one or more MHC constituentproteins, such as one or more HLA proteins and/or beta-2 microglobulin(B2M), in a cell, whereby rejection of non-autologous (e.g., allogeneic)cells by the recipient's immune system can be reduced or avoided. Inpreferred embodiments, one or more HLA class I proteins, such as HLA-A,B and/or C, and/or B2M may be knocked-out or knocked-down. Preferably,B2M may be knocked-out or knocked-down. By means of an example, Ren etal., (2017) Clin Cancer Res 23 (9) 2255-2266 performed lentiviraldelivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targetingendogenous TCR, β-2 microglobulin (B2M) and PD1 simultaneously, togenerate gene-disrupted allogeneic CAR T cells deficient of TCR, HLAclass I molecule and PD1.

In other embodiments, at least two genes are edited. Pairs of genes mayinclude, but are not limited to PD1 and TCRα, PD1 and TCRβ, CTLA-4 andTCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ,TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 andTCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 andTCRα, 2B4 and TCRβ.

In certain embodiments, a cell may be multiply edited (multiplex genomeediting) as taught herein to (1) knock-out or knock-down expression ofan endogenous TCR (for example, TRBC1, TRBC2 and/or TRAC), (2) knock-outor knock-down expression of an immune checkpoint protein or receptor(for example PD1, PD-L1 and/or CTLA4); and (3) knock-out or knock-downexpression of one or more MHC constituent proteins (for example, HLA-A,B and/or C, and/or B2M, preferably B2M).

Whether prior to or after genetic modification of the T cells, the Tcells can be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,232,566;7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631. Tcells can be expanded in vitro or in vivo.

Immune cells may be obtained using any method known in the art. In oneembodiment T cells that have infiltrated a tumor are isolated. T cellsmay be removed during surgery. T cells may be isolated after removal oftumor tissue by biopsy. T cells may be isolated by any means known inthe art. In one embodiment, the method may comprise obtaining a bulkpopulation of T cells from a tumor sample by any suitable method knownin the art. For example, a bulk population of T cells can be obtainedfrom a tumor sample by dissociating the tumor sample into a cellsuspension from which specific cell populations can be selected.Suitable methods of obtaining a bulk population of T cells may include,but are not limited to, any one or more of mechanically dissociating(e.g., mincing) the tumor, enzymatically dissociating (e.g., digesting)the tumor, and aspiration (e.g., as with a needle).

The bulk population of T cells obtained from a tumor sample may compriseany suitable type of T cell. Preferably, the bulk population of T cellsobtained from a tumor sample comprises tumor infiltrating lymphocytes(TILs).

The tumor sample may be obtained from any mammal. Unless statedotherwise, as used herein, the term “mammal” refers to any mammalincluding, but not limited to, mammals of the order Logomorpha, such asrabbits; the order Carnivora, including Felines (cats) and Canines(dogs); the order Artiodactyla, including Bovines (cows) and Swines(pigs); or of the order Perssodactyla, including Equines (horses). Themammals may be non-human primates, e.g., of the order Primates, Ceboids,or Simoids (monkeys) or of the order Anthropoids (humans and apes). Insome embodiments, the mammal may be a mammal of the order Rodentia, suchas mice and hamsters. Preferably, the mammal is a non-human primate or ahuman. An especially preferred mammal is the human.

T cells can be obtained from a number of sources, including peripheralblood mononuclear cells, bone marrow, lymph node tissue, spleen tissue,and tumors. In certain embodiments of the present invention, T cells canbe obtained from a unit of blood collected from a subject using anynumber of techniques known to the skilled artisan, such as Ficollseparation. In one preferred embodiment, cells from the circulatingblood of an individual are obtained by apheresis or leukapheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In one embodiment, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In one embodiment of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations. Initial activation steps in the absence of calciumlead to magnified activation. As those of ordinary skill in the artwould readily appreciate a washing step may be accomplished by methodsknown to those in the art, such as by using a semi-automated“flow-through” centrifuge (for example, the Cobe 2991 cell processor)according to the manufacturer's instructions. After washing, the cellsmay be resuspended in a variety of biocompatible buffers, such as, forexample, Ca-free, Mg-free PBS. Alternatively, the undesirable componentsof the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient. A specificsubpopulation of T cells, such as CD28+, CD4+, CDC, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selectiontechniques. For example, in one preferred embodiment, T cells areisolated by incubation with anti-CD3/anti-CD28 (i.e., 3×28)-conjugatedbeads, such as DYNABEADS® M-450 CD3/CD28 T, or XCYTE DYNABEADS™ for atime period sufficient for positive selection of the desired T cells. Inone embodiment, the time period is about 30 minutes. In a furtherembodiment, the time period ranges from 30 minutes to 36 hours or longerand all integer values there between. In a further embodiment, the timeperiod is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferredembodiment, the time period is 10 to 24 hours. In one preferredembodiment, the incubation time period is 24 hours. For isolation of Tcells from patients with leukemia, use of longer incubation times, suchas 24 hours, can increase cell yield. Longer incubation times may beused to isolate T cells in any situation where there are few T cells ascompared to other cell types, such in isolating tumor infiltratinglymphocytes (TIL) from tumor tissue or from immunocompromisedindividuals. Further, use of longer incubation times can increase theefficiency of capture of CD8+ T cells.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. A preferred method iscell sorting and/or selection via negative magnetic immunoadherence orflow cytometry that uses a cocktail of monoclonal antibodies directed tocell surface markers present on the cells negatively selected. Forexample, to enrich for CD4+ cells by negative selection, a monoclonalantibody cocktail typically includes antibodies to CD14, CD20, CD11b,CD16, HLA-DR, and CD8.

Further, monocyte populations (i.e., CD14+ cells) may be depleted fromblood preparations by a variety of methodologies, including anti-CD14coated beads or columns, or utilization of the phagocytotic activity ofthese cells to facilitate removal. Accordingly, in one embodiment, theinvention uses paramagnetic particles of a size sufficient to beengulfed by phagocytotic monocytes. In certain embodiments, theparamagnetic particles are commercially available beads, for example,those produced by Life Technologies under the trade name Dynabeads™. Inone embodiment, other non-specific cells are removed by coating theparamagnetic particles with “irrelevant” proteins (e.g., serum proteinsor antibodies). Irrelevant proteins and antibodies include thoseproteins and antibodies or fragments thereof that do not specificallytarget the T cells to be isolated. In certain embodiments the irrelevantbeads include beads coated with sheep anti-mouse antibodies, goatanti-mouse antibodies, and human serum albumin.

In brief, such depletion of monocytes is performed by preincubating Tcells isolated from whole blood, apheresed peripheral blood, or tumorswith one or more varieties of irrelevant or non-antibody coupledparamagnetic particles at any amount that allows for removal ofmonocytes (approximately a 20:1 bead:cell ratio) for about 30 minutes to2 hours at 22 to 37 degrees C., followed by magnetic removal of cellswhich have attached to or engulfed the paramagnetic particles. Suchseparation can be performed using standard methods available in the art.For example, any magnetic separation methodology may be used including avariety of which are commercially available, (e.g., DYNAL® MagneticParticle Concentrator (DYNAL MPC®)). Assurance of requisite depletioncan be monitored by a variety of methodologies known to those ofordinary skill in the art, including flow cytometric analysis of CD14positive cells, before and after depletion.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8+ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4+ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8+ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×106/ml. In other embodiments, theconcentration used can be from about 1×105/ml to 1×106/ml, and anyinteger value in between.

T cells can also be frozen. Wishing not to be bound by theory, thefreeze and subsequent thaw step provides a more uniform product byremoving granulocytes and to some extent monocytes in the cellpopulation. After a washing step to remove plasma and platelets, thecells may be suspended in a freezing solution. While many freezingsolutions and parameters are known in the art and will be useful in thiscontext, one method involves using PBS containing 20% DMSO and 8% humanserum albumin, or other suitable cell freezing media, the cells then arefrozen to −80° C. at a rate of 1° per minute and stored in the vaporphase of a liquid nitrogen storage tank. Other methods of controlledfreezing may be used as well as uncontrolled freezing immediately at−20° C. or in liquid nitrogen.

T cells for use in the present invention may also be antigen-specific Tcells. For example, tumor-specific T cells can be used. In certainembodiments, antigen-specific T cells can be isolated from a patient ofinterest, such as a patient afflicted with a cancer or an infectiousdisease. In one embodiment neoepitopes are determined for a subject andT cells specific to these antigens are isolated. Antigen-specific cellsfor use in expansion may also be generated in vitro using any number ofmethods known in the art, for example, as described in U.S. PatentPublication No. US 20040224402 entitled, Generation and Isolation ofAntigen-Specific T Cells, or in U.S. Pat. No. 6,040,177.Antigen-specific cells for use in the present invention may also begenerated using any number of methods known in the art, for example, asdescribed in Current Protocols in Immunology, or Current Protocols inCell Biology, both published by John Wiley & Sons, Inc., Boston, Mass.

In a related embodiment, it may be desirable to sort or otherwisepositively select (e.g. via magnetic selection) the antigen specificcells prior to or following one or two rounds of expansion. Sorting orpositively selecting antigen-specific cells can be carried out usingpeptide-MHC tetramers (Altman, et al., Science. 1996 Oct. 4;274(5284):94-6). In another embodiment the adaptable tetramer technologyapproach is used (Andersen et al., 2012 Nat Protoc. 7:891-902).Tetramers are limited by the need to utilize predicted binding peptidesbased on prior hypotheses, and the restriction to specific HLAs.Peptide-MHC tetramers can be generated using techniques known in the artand can be made with any MEW molecule of interest and any antigen ofinterest as described herein. Specific epitopes to be used in thiscontext can be identified using numerous assays known in the art. Forexample, the ability of a polypeptide to bind to MEW class I may beevaluated indirectly by monitoring the ability to promote incorporationof 125I labeled β2-microglobulin (β2m) into MEW class I/β2m/peptideheterotrimeric complexes (see Parker et al., J. Immunol. 152:163, 1994).

In one embodiment cells are directly labeled with an epitope-specificreagent for isolation by flow cytometry followed by characterization ofphenotype and TCRs. In one T cells are isolated by contacting the T cellspecific antibodies. Sorting of antigen-specific T cells, or generallyany cells of the present invention, can be carried out using any of avariety of commercially available cell sorters, including, but notlimited to, MoFlo sorter (DakoCytomation, Fort Collins, Colo.),FACSAria™, FACSArray™, FACSVantage™, BD™ LSR II, and FACSCalibur™ (BDBiosciences, San Jose, Calif.).

In a preferred embodiment, the method comprises selecting cells thatalso express CD3. The method may comprise specifically selecting thecells in any suitable manner. Preferably, the selecting is carried outusing flow cytometry. The flow cytometry may be carried out using anysuitable method known in the art. The flow cytometry may employ anysuitable antibodies and stains. Preferably, the antibody is chosen suchthat it specifically recognizes and binds to the particular biomarkerbeing selected. For example, the specific selection of CD3, CD8, TIM-3,LAG-3, 4-1BB, or PD-1 may be carried out using anti-CD3, anti-CD8,anti-TIM-3, anti-LAG-3, anti-4-1BB, or anti-PD-1 antibodies,respectively. The antibody or antibodies may be conjugated to a bead(e.g., a magnetic bead) or to a fluorochrome. Preferably, the flowcytometry is fluorescence-activated cell sorting (FACS). TCRs expressedon T cells can be selected based on reactivity to autologous tumors.Additionally, T cells that are reactive to tumors can be selected forbased on markers using the methods described in patent publication Nos.WO2014133567 and WO2014133568, herein incorporated by reference in theirentirety. Additionally, activated T cells can be selected for based onsurface expression of CD107a.

In one embodiment of the invention, the method further comprisesexpanding the numbers of T cells in the enriched cell population. Suchmethods are described in U.S. Pat. No. 8,637,307 and is hereinincorporated by reference in its entirety. The numbers of T cells may beincreased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), morepreferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-,or 90-fold), more preferably at least about 100-fold, more preferably atleast about 1,000 fold, or most preferably at least about 100,000-fold.The numbers of T cells may be expanded using any suitable method knownin the art. Exemplary methods of expanding the numbers of cells aredescribed in patent publication No. WO 2003057171, U.S. Pat. No.8,034,334, and U.S. Patent Application Publication No. 2012/0244133,each of which is incorporated herein by reference.

In one embodiment, ex vivo T cell expansion can be performed byisolation of T cells and subsequent stimulation or activation followedby further expansion. In one embodiment of the invention, the T cellsmay be stimulated or activated by a single agent. In another embodiment,T cells are stimulated or activated with two agents, one that induces aprimary signal and a second that is a co-stimulatory signal. Ligandsuseful for stimulating a single signal or stimulating a primary signaland an accessory molecule that stimulates a second signal may be used insoluble form. Ligands may be attached to the surface of a cell, to anEngineered Multivalent Signaling Platform (EMSP), or immobilized on asurface. In a preferred embodiment both primary and secondary agents areco-immobilized on a surface, for example a bead or a cell. In oneembodiment, the molecule providing the primary activation signal may bea CD3 ligand, and the co-stimulatory molecule may be a CD28 ligand or4-1BB ligand.

In certain embodiments, T cells comprising a CAR or an exogenous TCR,may be manufactured as described in WO2015120096, by a methodcomprising: enriching a population of lymphocytes obtained from a donorsubject; stimulating the population of lymphocytes with one or moreT-cell stimulating agents to produce a population of activated T cells,wherein the stimulation is performed in a closed system using serum-freeculture medium; transducing the population of activated T cells with aviral vector comprising a nucleic acid molecule which encodes the CAR orTCR, using a single cycle transduction to produce a population oftransduced T cells, wherein the transduction is performed in a closedsystem using serum-free culture medium; and expanding the population oftransduced T cells for a predetermined time to produce a population ofengineered T cells, wherein the expansion is performed in a closedsystem using serum-free culture medium. In certain embodiments, T cellscomprising a CAR or an exogenous TCR, may be manufactured as describedin WO2015120096, by a method comprising: obtaining a population oflymphocytes; stimulating the population of lymphocytes with one or morestimulating agents to produce a population of activated T cells, whereinthe stimulation is performed in a closed system using serum-free culturemedium; transducing the population of activated T cells with a viralvector comprising a nucleic acid molecule which encodes the CAR or TCR,using at least one cycle transduction to produce a population oftransduced T cells, wherein the transduction is performed in a closedsystem using serum-free culture medium; and expanding the population oftransduced T cells to produce a population of engineered T cells,wherein the expansion is performed in a closed system using serum-freeculture medium. The predetermined time for expanding the population oftransduced T cells may be 3 days. The time from enriching the populationof lymphocytes to producing the engineered T cells may be 6 days. Theclosed system may be a closed bag system. Further provided is populationof T cells comprising a CAR or an exogenous TCR obtainable or obtainedby said method, and a pharmaceutical composition comprising such cells.

In certain embodiments, T cell maturation or differentiation in vitromay be delayed or inhibited by the method as described in WO2017070395,comprising contacting one or more T cells from a subject in need of a Tcell therapy with an AKT inhibitor (such as, e.g., one or a combinationof two or more AKT inhibitors disclosed in claim 8 of WO2017070395) andat least one of exogenous Interleukin-7 (IL-7) and exogenousInterleukin-15 (IL-15), wherein the resulting T cells exhibit delayedmaturation or differentiation, and/or wherein the resulting T cellsexhibit improved T cell function (such as, e.g., increased T cellproliferation; increased cytokine production; and/or increased cytolyticactivity) relative to a T cell function of a T cell cultured in theabsence of an AKT inhibitor.

In certain embodiments, a patient in need of a T cell therapy may beconditioned by a method as described in WO2016191756 comprisingadministering to the patient a dose of cyclophosphamide between 200mg/m2/day and 2000 mg/m2/day and a dose of fludarabine between 20mg/m2/day and 900 mg/m²/day.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1—Charting the Ascites Ecosystem by scRNA-Seq

A translational workflow was adopted from previous work (Tirosh et al.,2016 Science, 352:189-196; B. Izar and A. Rotem, 2016 Curr. Protoc. Mol.Biol., 116:28.8.1-28.8.12, FIG. 18) for scRNA-seq (FIG. 17 illustratesrationale for single-cell sequencing compared to bulk sequencing) ofascites (FIG. 15 illustrates ascites in the female reproductive system)from patients with HGSC. Upon drainage of ascites, fresh specimens wereimmediately processed by removal of red blood cells (RBCs), isolation ofa cell pellet, depletion of CD45+ immune cells (a major component ofascites) and scRNA-seq profiling using the 10× Genomics dropletplatform. Eight specimens were profiled, and following quality controlsstudies included single-cell profiles for further analysis. The numberof cells profiled per patient, their prior treatments, baselinedemographics and BRCA1/2 mutation status were determined. Notably, fortwo patients (3250 and 3288), specimens were collected at two differenttime points, and in one of those cases (3288) the first specimen wascollected prior to any therapy, and the second (named 3288.1) wascollected after the first cycle of treatment with carboplatin pluspaclitaxel.

A t-distributed stochastic neighbor embedding (tSNE) analysis wasperformed, which revealed 20 distinct cell clusters (FIG. 1A and FIG.1B). Clusters were annotated based on the top differentially expressedgenes as either epithelial (e.g. by epithelial cell adhesion molecule(EPCAM) and cytokeratins and kallikreins), macrophages (e.g. by clusterof differentiation (CD)14, allograft inflammatory factor (AIF)1, colonystimulating factor 1 receptor (CSF1R) and CD163) fibroblasts (e.g. bypodoplanin (PDPN), decorin (DCN) and THY1 (CD90)), dendritic cells (e.g.by CD1C, CD1E, C-C motif chemokine receptor (CCR)7 and CD83), B cells(e.g. by CD19 and CD79A/B), T cells (e.g. by CD2 and CD3D/E/G) anderythrocytes (e.g. by GATA binding protein (GATA)1 and hemoglobin) (FIG.1C). Even though studies depleted immune cells, the cellular compositionwas still dominated by immune cells and in particular by macrophages.Nevertheless, there was extensive variability in the proportions of celltypes and clusters among patients (FIG. 1B). For example, in threepatients (3281, 3288 and 3290), the most abundant cell type wasmacrophages, in two patients it was epithelial cells and in one patient(3266) it was B cells. Some of these differences relate to differentialefficiency in immune depletion or in cellular viability, yet otherdifferences are more likely biological, such as the relative proportionof distinct immune cells (e.g. macrophage vs. lymphocytes) or non-immunecells (e.g. epithelial vs. fibroblasts).

After gleaning initial insights into the main cell types that constitutemalignant ascites, studies were completed to resolve the diversity ofcellular states that correspond to each cell type, including the fourclusters of epithelial cells, four clusters of fibroblasts, fourclusters of macrophages and two clusters of dendritic cells. For eachcell type, differentially expressed genes between distinct clusters wereidentified (FIG. 1D). For example, the malignant cell clusters differedby the expression of GATA2, SRY (sex determining region Y)-box 2 (SOX2),claudin (CLDN)6/9, homeobox protein (HOX)B5 and other genes (FIG. 1D).

Within the immune cell types, analysis of differentially expressed genessuggested that the two dendritic clusters differ by their maturation,with immature CD83-high dendritic cells (DCs) and more mature CCR7-highcells. The four macrophage clusters differed in the expression ofvarious genes including those involved in activation (e.g. FBJ murineosteosarcoma viral oncogene homolog B (FOSB)), proliferation (e.g.cyclin-dependent kinase (CDK)1) and immune suppression (indoleamine2,3-dioxygenase 1 (IDO1)).

Interestingly, the fibroblasts demonstrated variability amongsub-groups; the four clusters showed high expression of definingmarkers, including CD90 (THY1), DCN, connective tissue growth factor(CTGF), as well as several complement factors (complement component(C)1R, C1S, complement factor (CF)I), in line with prior observations inmelanoma (Tirosh et al., 2016 Science, 352:189-196, FIG. 1C). However,the fibroblast clusters differed by the expression of additionalcomplement factors (C1QA/B/C, CFB), chemokines (CXCL1/2/10/12), andcytokines (IL6 and IL10) (FIG. 1D). These immune-related genes wereconsistently higher in two of the fibroblasts clusters, suggesting thatthese reflect immunomodulatory fibroblasts (P. Cirri and P. Chiarugi,2011 Am. J. Cancer Res., 1:482-497). Notably, CXCL12, the ligand forCXCR4 on ovarian cancer cells, was highly expressed by all fibroblastpopulations. The CXCL12-CXCR4 axis was shown to activate STAT3 signalingand driver metastasis in breast and lung cancer (X. Liu et al., 2014Oncol. Rep., 32:2760-2768; M. Pfeiffer et al., 2009 Br. J. Cancer,100:1949-1956), and this interaction has been therapeutically exploited(Kajiyama et al., 2008 Int. J. Cancer, 122:91-99).

IL-6, a cytokine with pleiotropic effects and the primary ligand thatactivates the JAK/STAT pathway (Darnell et al., 1994 Science,264:1415-1421), constitutes a key cytokine in ovarian cancer ascites(Matte et al., 2012 Am. J. Cancer Res., 2:566-580), links inflammationto cancer (Iliopoulos et al., 2009 Cell, 139:693-706; Kulbe et al., 2012Cancer Res., 72:66-75) and is associated with a poor prognosis (Cowardet al., 2011 Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res.,17:6083-6096). Most cells in the ascites ecosystem express IL6, butexpression was particularly high by two fibroblast clusters 8 and 9.Thus, subsets of fibroblasts express distinct immunomodulatory programsthat shape the immune environment in ascites.

Example 2—scRNA-Seq of Cancer Cells Reveals Inter- and Intra-TumorHeterogeneity in HGSC

The droplet-based profiling provided insights into transcriptomes of alarge number of cells, but had a relatively low number of cancer cellsand with limited coverage of each single cell transcriptome. To moredeeply interrogate malignant cells isolated from ascites, studies werecompleted with enriching specifically for cancer cells. For thispurpose, viable cancer cells were isolated by fluorophore-activated cellsorting (FACS) in 96-well plates staining for EPCAM and CD24. Thecombination of these surface markers was previously shown to be highlysensitive and specific for ovarian cancer cells (Peterson et al., 2013Proc. Natl. Acad. Sci. U.S.A., 110:E4978-4986). Individually sortedcells were profiled using the a modified Smart-Seq2 protocol (Picelli etal., 2013 Nat. Methods, 10:1096-1098; Tombetta et al., 2014 Curr.Protoc. Mol. Biol., 107:4.22.1-4.22.17). With this approach,significantly greater cancer cell proportions were obtained (see belowalong with higher transcriptome coverage). Fourteen specimens werecollected and profiles passing quality control assessment were obtained.

In FIG. 2A-FIG. 2E using the same experimental design as FIG. 1A-FIG.1D, tSNE was used to find that tumor cells cluster mostly based on theirtumor of origin. Again, data find an overrepresentation of inflammatorypathways, including TNF-alpha signaling and STAT-3 signaling in theseresistant patient single-cells.

Eight clusters were identified by tSNE and density clustering (FIG. 2Aand FIG. 2B). Differential expression across these clusters (FIG. 2C)indicated that the vast majority of cells were epithelial (clusters1-6), while a small proportion of cells were fibroblasts (cluster 7) andmacrophages (cluster 8). The cancer cells clustered primarily by theirpatient of origin (FIG. 2B), with inter-individual heterogeneity in theexpression of many genes (FIG. 2B). For example, cluster 1 (mappingprimarily to patient GL10) showed significantly higher expression ofCD133, a cell surface marker frequently used to identify putativeovarian cancer stem cells. Other variable genes among the epithelialclusters included IL6 and CXCL3, indicating cancer cell-intrinsicinflammatory signaling.

To further examine this inter-tumor heterogeneity, cells were restrictedfrom each cluster to those from the dominant patient while excluding thefew cells that map to cluster of a different patient. To validate thatthese epithelial clusters correspond to malignant cells, chromosomalcopy number variations (CNVs) were inferred from the average expressionof genes in each chromosomal region (FIG. 2D). As expected, asubstantial burden of CNVs across the genomic landscape was found, whichwas not detected in a control analysis where the genes were randomlyassigned to genomic locations (FIG. 2D), Using this approach, themajority of CNVs identified by whole-exome sequencing of 489 high-gradeserous ovarian cancers were identified (T. C. G. A. R. Network, 2011Nature, 474:609-615). While the resolution of this approach isrestricted to inference of large-scale CNVs, CNVs were not detected thatwere exclusive to one cluster, indicating that variability in geneexpression among clusters was not primarily due to genomic differencesamong patients.

To investigate whether TCGA-identified subtypes (differentiated,proliferative, mesenchymal and immunoreactive) explain differencesbetween patients, each cluster was scored for the expression ofTCGA-derived subtype signatures. All six cancer cell clusters highlyexpressed the “differentiated” signature and only one cluster (cluster4) strongly expressed the “proliferative” signature (FIG. 2E).Surprisingly, the “mesenchymal” and “immunoreactive” signatures showedweak or no expression in cancer cell clusters and were only reflectiveof fibroblast infiltration and immune cell infiltration, and notintrinsic to the tumor cells. The mesenchymal signature was primarilyreflective of fibroblast gene expression, while the immunoreactivesignature was primarily expressed by macrophages. These results suggestthat (i) the mesenchymal and immunoreactive subtypes previouslyidentified by bulk RNA-profiling probably represent the intratumoralabundance of fibroblasts and macrophages, respectively, in the tumormicroenvironment; (ii) the differentiated subtype reflects a consistentprogram of cancer cells across HGSOC, and thus mapped to tumors of highpurity; (T. C. G. A. R. Network, 2011 Nature, 474:609-615) theproliferative subtype is the subtype that truly reflects a uniqueprogram that is specific to a subset of HGSC tumors.

Example 3—Unbiased Analysis of Intra-Tumor Heterogeneity RevealsExpression of Distinct Inflammatory Programs and a Shared Activation ofthe JAK/STAT Pathway in Platinum-Resistant Ovarian Cancer

FIG. 16A-FIG. 16C depict a series of bar charts and gene expressionprofiles illustrating previous genomic characterization of ovariancancer and platinum-resistance (Tirosh et al., 2016 Science.,352:189-196; Gaillard et al., 2016 Gynecology Oncol. Res. Pract., 3:11),FIG. 14 illustrates ideal ovarian cancer therapy.

The next study completed assessed patterns of intra-tumor heterogeneityin five patients for which single-cell RNA-seq profiles of >100 cancercells were obtained. For each patient, non-negative matrix factorization(NMF) was used to identify expression modules that consist of genes withcoherent variability across cancer cells and highlight a subset ofcells. Identified were 6-9 such modules in each tumor for a total of 35modules (FIG. 3A-FIG. 3C, FIG. 10). Examining the top genes thatcorrespond to each of these modules suggested diverse functions,including cell cycle (e.g. cyclin-A (CCNA)2, CCNB2, aurora kinase B(AURKB)), inflammation (e.g. IL6, IL32, TNF, interferon alpha inducibleprotein (IFI)6, and stress or activation (e.g. heat shock protein familyA (HSPA)5-7, activating transcription factor (ATF)4, c-Jun Kinase (JNK),DNA damage-inducible transcript 3 (DDIT3)).

The top genes of module #1 from GL10 included ALDH1A3 and CD133 (PROM1)(FIG. 8A). The consistent expression of these prominent stem-nessmarkers (Silva et al., 2011 Cancer Res., 71:3991-4001) suggests thatthis module highlights a subset of ovarian cancer stem cells. Other topgenes in this module are associated with mesenchymal phenotypes(fibronectin (FN)1, alpha-actin 2 (ACTA2) and myosin light chain gene(MYL9)) and secreted molecules, such as growth arrest specific protein 6(GAS6), the only known ligand for the tyrosine kinase AXL, which isimplicated in de-differentiation and drug resistance in several cancers(X. Wu et al., 2014 Oncotarget, 5:9546-9563). However, similar moduleswere not detected in any of the other four patients. As another approachto evaluate the presence of stem-ness programs, studies were performedthat tested, in each tumor, the correlation among these three stem-nessmarkers. Consistent with the NMF analysis, the stem-ness markers weresignificantly (P<0.05) correlated only in GL10. Furthermore, the genesmost correlated with these stem-ness markers in GL10 were notco-expressed in other tumors (FIG. 8D-FIG. 8E). Taken together, aputative stem-ness program in GL10 was identified, but profiling oflarger patient cohorts is required to examine whether this reflects arecurrent signature in ovarian cancer.

The study next examined whether there are consistent expression programsthat functionally relate different tumors to each other. To this end,the overlap between the top genes for all pairs of modules definedacross the five patients were examined (FIG. 3D). The most significantassociation among tumors was the expression of a cell cycle program(FIG. 3E), which is expected given that each tumor contains both cyclingand non-cycling cells and the cell cycle programs are highly robust.Furthermore, programs dominated by immune- or inflammation-associatedgenes were found to be shared among two or more patients (FIG. 3E).These cancer cell expressed modules included inflammatory cytokines(e.g. IL-6, TNF, IL-8, IL-32), antigen presentation through majorhistocompatibility complex (MHC) class II (e.g. CD74 and HLA class IIhistocompatibility antigen, DR alpha chain (HLA-DRA)) and interferonresponse (e.g. IFI6, IFIT1, interferon-stimulated gene (ISG)15). Acommon feature across the vast majority of cells from platinum-resistantpatients was the convergence of several of these cytokines and pathwaysonto the JAK/STAT pathway. Indeed, several components of the JAK/STATpathway, including JAK1, STAT3, STAT2, STAT1, and oncostatin M receptor(OSMR) were found to be among the most highly expressed genes inplatinum-resistant patients. OSMR is a recently describedSTAT3-regulated cytokine receptor that shares a downstreamtranscriptional network with STAT3 (FIG. 3F). Among these genes, JAK1and STAT3 demonstrated strong expression above the expected within thecancer cell population, indicating that signaling occurred primarily viathe JAK/STAT3 pathway, indicating the role of JAK/STAT3 pathway inplatinum-resistance.

For three of the five patients described above (GL10, GL13 and GL15),samples were collected successfully with high quality single-celltranscriptomes at multiple time points. In these patients, significantdifferences of expression profiles between the different time pointswere not detected (FIG. 8A-FIG. 8E). This observation is in line withprior studies showing lack of acquired genomic aberrations in matchedsamples, but is likely, in part, explained by the timing of samplecollection, which occurred in a relatively rapid succession (over weeks)without drastic changes in therapy. However, this experiment highlightsthe clinical feasibility of collecting several samples from the samepatient and, when timed appropriately, provides insights into theevolution of cell heterogeneity. To gain insights into changes thatoccur during therapy and at the time of treatment resistance,patient-derived xenograft (PDX) models were used.

Example 4—Longitudinal Analysis of HGSC Expression Profiles in PDXModels

Next, platinum-resistance was modeled using PDX-models. For thispurpose, three previously established PDX models of ovarian cancergenerated from patient-derived ovarian cancer ascites serially passagedin immunodeficient mice were used, including one BRCA-WT (DF20) and twoBRCA-mutant (DF68 and DF101) models (Liu et al., 2016 Clin. Cancer Res.,23(5):1263-1273). These PDX models mimic the clinical course ofpatients, including initial response to platinum-based therapies andtumor regression that is followed by tumor relapse weeks to months uponcompletion of therapy (Liu et al., 2016 Clin. Cancer Res.,23(5):1263-1273) (FIG. 4A). As previously described (Liu et al., 2016Clin. Cancer Res., 23(5):1263-1273), patient-derived cells were stablytransfected with luciferase and mCherry, enabling non-invasivemonitoring by in vivo bioluminescence imaging (BLI), and subsequentisolation of individual cells (flow-sorting for mCherry-expressingcells). PDX models developed tumors within 3 weeks followingsubcutaneous implantation and were treated with vehicle or carboplatin.Single-cells were harvested for sequencing from untreated animals(vehicle) and the carboplatin-treated animals were monitored for tumorregression and relapse. As expected, there was a brisk response tocarboplatin (indicated by reduced tumor volume and decreased BLI signal)followed by subsequent tumor outgrowth (FIG. 4A). Single-cells werecollected from one cohort of animals at the time of maximal response andminimal residual disease (MRD), and from another cohort of animals atthe time they developed fully relapsed tumors (relapse). In total,scRNAseq was performed on ˜800 single-cells isolated from 5 vehicle, 6MRD and 8 relapsed animals. Global comparison of individual cells oraverage profiles (FIG. 4B) from these 19 mice demonstrated that theycluster based on their parental PDX-model with high similarity acrossexperimental groups (i.e. vehicle, MRD and relapse samples). The studyfocused primarily on DF20 and profiled multiple mice for eachexperimental group (3 vehicle, 4 MRD and 4 relapse mice) and found thatthese 11 samples largely clustered by the model of origin and not byexperimental group (FIG. 4B). To examine the possibility of morerestricted changes in gene expression, profiles of MRD or relapseanimals were compared to the vehicle animals of the same model anddefined differentially expressed genes. The study identified upregulatedgenes in each model, yet their overlap across models was minimal (FIG.4C), arguing against a consistent program of tumor relapse. Across allmodels, strong expression of key nodes of the JAK/STAT pathway werefound, including JAK1 and STAT3 (FIG. 4D). Furthermore, the study foundthat STAT1, fibroblast growth factor receptor (FGFR)1 and CXCR4 (thereceptor for CXCL-12-mediated JAT/STAT-activation) were more stronglyexpressed in the MRD/relapse samples compared to vehicle treated cells,further supporting the notion of a role of the JAK/STAT pathway inplatinum-resistance (FIG. 9). Accordingly, gene sets were detected whichwere consistently altered across two or three samples of MRD (FIG. 4D)or relapse (FIG. 4E), and contribute to drug resistance or tumorrelapse. These include several chemokines (CXCL2/10/11), genes (HLA-DRA,and HLA class II histocompatibility antigen, DR beta chain (HLA-DRB)1,HLA class II histocompatibility antigen, DQ beta chain 1(HLA-DQB1)), theimmune-suppression gene IDO1, a MYB-like transcription factor (MYBL2)which was previously associated with poor survival in breast cancer(Amatschek et al., 2004 Cancer Res., 64:844-856) and the growth factorFGF13 which was recently implicated in cancer cell survival (Bublik etal., 2017 Proc. Natl. Acad. Sci., 114:E496-E505) and in resistance tocisplatin (Okada et al., 2013 Sci. Rep., 3:2899).

Example 5—Intra-Tumor Heterogeneity in PDX Models

The study next examined the intra-tumor variability of cell states amongindividual mice and as described above for patient samples, NMF was usedto identify expression modules with high variability in each of thethree PDX models (FIG. 5A-FIG. 5C). PDX modules were then compared tothe patient modules (FIG. 5D). As expected, the highest similarity wasobserved for cell cycle modules (PDX modules 1-3) (FIG. 5D and FIG. 5E).Of the three immune-related programs described in the patient analysis,the interferon program was also identified in the PDX analysis (PDXmodules 6-8) (FIG. 5E). The other two immune-related programs (thecytokine and modules) found in patients were not strongly expressed inPDX models (FIG. 10), indicating that extracellular immune cell mediatedstimuli, which are mostly absent in these models, contribute to theexpression of these programs.

While cross-comparison between platinum-resistant patients andPDX-models did not identify a single mechanism of drug resistance, aconsistent observation gleaned from single-cell analyses was thesignificant cancer cell-autonomous expression of cell cycle modules,inflammatory and other immune related pathways, in particular expressionof key JAK/STAT-pathway nodes and controlled pathways, includinginterferon signaling, MEW expression, interferon response elements andother cytokine markers. This notion was further supported by theexpression of activators of JAK/STAT signaling by non-malignant cells,including IL6, CXCL12-CXCR4 interactions and other cytokines.Collectively, the scRNA-seq data highlighted the importance of JAK/STATactivation in ovarian cancer via both autocrine (tumor-cell intrinsic)and paracrine (tumor cell microenvironment) mechanisms. Suggesting arole for this pathway in ovarian cancer cell survival, pathophysiologyin metastasis and platinum-resistance (FIG. 22 depicts immune cells andJAK/STAT expression in platinum-resistant ovarian cancer cells). TheJAK/STAT pathway and its upstream activators (e.g. IL6, TNF, IFN,CXCL12) have been previously implicated in ovarian cancer cell survival,metastasis, angiogenesis, and chemo-resistance (Kulbe et al., 2012Cancer Res., 72:66-75; Coward et al., 2011 Clin. Cancer Res. Off. J. Am.Assoc. Cancer Res., 17:6083-6096; Wen et al., 2014 Mol. Cancer Ther.13:3037-3048; Duan et al., 2006 Clin. Cancer Res. Off. J. Am. Assoc.Cancer Res., 12:5055-5063; Saini et al., 2017 Oncogene, 36:168-181;Stone et al., 2012 N. Engl. J. Med., 366:610-618) and the studyestablishes the significance of this pathway in ovarian cancer at thesingle-cell level.

Example 6—Inhibition of JAK/STAT Signaling Inhibits Spheroid Formation,Spheroid Integrity, and Invasiveness Though a Mesothelial Monolayer andLeads to Disruption of Formed Spheroids

Inhibition of the JAK/STAT pathway, represents a feasible therapeuticavenue for ovarian cancer, including chemo-resistant disease.Two-dimensional and three-dimensional cell cultures of two commonly usedovarian cancer cell lines were screened, OVACR4 and OVACR8 with alibrary of 14 compounds inhibiting different nodes of the JAK/STATpathway (FIG. 6A, FIG. 11A-FIG. 11D). This initial screen identifiedJSI-124, a previously characterized selective JAK/STAT inhibitor(Blaskovich et al., 2003 Cancer Res., 63:1270-1279). The on-targetactivity of JSI-124 was confirmed using luciferase assay for STAT3activity in oncostatin M (OSM) stimulated Heya8 cell lines (FIG. 11E).

To test whether JSI-124 overcomes platinum-resistance in patient-models,ex vivo cell cultures of three platinum-resistant patients (DF3266,DF3288, DF3291) were generated. Single-cell RNA-sequencing was performedon these platinum-resistant patient samples, using the growth in lowattachment (GILA) assay (Rotem et al., 2015 Proc. Natl. Acad. Sci.U.S.A., 112:5708-5713; B. Izar and A. Rotem, 2016 Curr. Protoc. Mol.Biol., 116:28.8.1-28.8.12). These ex vivo cultures were treated withJSI-124, a previously characterized highly selective JAK/STAT3 inhibitor(Blaskovich et al., 2003 Cancer Res., 63:1270-1279), and the efficacy ofJSI-124 was compared to drugs that are most widely used for thetreatment of ovarian cancer, including carboplatin, cisplatin,paclitaxel, and olaparib, a PARP-inhibitor. In all tested models, strongsingle-agent cytotoxic activity of JSI-124 with EC₅₀ values in nanomolarranges, and an E_(max) of up to 99% were observed (FIG. 6B). Incontrast, all spheroid cultures were resistant to carboplatin,cisplatin, olaparib and paclitaxel at a similar dose range, with theexception of cisplatin with an EC₅₀ of ˜10 μM in patients DF3266 andDF3288. Microscopic analysis of spheroids showed that while othertherapies changed the morphology of spheroids, only JSI-124 led todisintegration of the solid 3D spheroid structure (FIG. 6C) that iscritical for invasive potential. The activity of JSI-124 against bothformed spheroids and conventional cell culture conditions were confirmedin additional established ovarian cancer cell lines from the Cancer CellLine Encyclopedia (CCLE)(J. Barretina et al., 2012 Nature, 483:603-607,FIG. 12). FIG. 36 depicts a series of photomicrographs showingmorphological description of ex-vivo patient-derived ovarian cells aftertreatment with STAT3 inhibitor JSI-124. FIG. 12A and FIG. 12B are aseries of graphs and FIG. 23 is a series of images further supportingthat JSI-124 effectively kills ovarian cancer cell lines grown astwo-dimensional cultures or three-dimensional spheroids. Since theformation of spheroids is a critical step in abdominal metastasis inovarian cancer, tests to confirm whether JSI-124 inhibited spheroidformation in vitro were completed. Compared to DMSO control, treatmentwith JSI-124 completely abrogated the formation of spheroids in fourcell lines ovarian cancer cell lines in low adherent surface cultureconditions, including OVCAR4, OVCAR8, OVASHO, and TYKNU, andsignificantly reduced the number of spheroids in one cell line(Kuramachi) (FIG. 6D). Importantly, this effect was not due to cellkilling by JSI-124. In contrast, carboplatin modest to no effects onspheroids formation at the same treatment doses (FIG. 6C).

Once spheroids are formed in the ascites ecosystem, they serve as avehicle for malignant cells to reach intra-abdominal sites ofmetastases. Because abdominal organs are covered with a mesothelial celllining, a critical step in metastasis is invasion (or clearance) throughthis cellular monolayer (Iwanicki et al., 2011 Cancer Discov.,1:144-157; Davidowitz et al., 2014 J. Clin. Invest., 124:2611-2625;Iwanicki et al., 20016 JCI Insight, 1). To test whether JSI-124 alsoinhibits invasion, a previously established system was used to determinethe ability to clear a mesothelial monolayer (Iwanicki et al., 2011Cancer Discov., 1:144-157). For this purpose, the ability of patientascites-derived spheroids to invade mesothelium with and withoutpre-treatment with JSI-124 for either 30 or 120 minutes (followed bydrug removal was measured. While untreated patient-derived spheroidsshowed a strong potential to clear the mesothelial monolayer, spheroidstreated with JSI-124 for 30 minutes or 120 minutes (followed by drugremoval) had a significantly reduced ability to invade the mesothelialmonolayer (FIG. 6E, FIG. 37). Importantly, this effect was not explainedby JSI-124 mediated disintegration of spheroids (FIG. 23 top row). Thisexperiment was repeated with two commonly used established ovariancancer cell lines (OVCAR8 and TKYNU) and found that inhibition withJSI-124 abolished their ability to invade the mesothelium (FIG. 6F andFIG. 6G, FIG. 23 bottom row) Together, these results indicate thatJSI-124 effectively disintegrates platinum-resistant ovarian cancerspheroids, inhibits spheroids formation and invasive properties, andthereby inhibits key steps of abdominal metastasis in ovarian cancer,even in tumors resistant to standard chemotherapeutics.

Example 7—Intraperitoneally Administered JSI-124 Prevents the AbdominalDevelopment of Malignant Ascites

Given that abdominal metastasis mediated by spheroids represents a keyclinical problem in patients with ovarian cancer (FIG. 13 illustratesthat 75% of ovarian cancers are diagnosed at stage III/IV), studies werecompleted to determine whether STAT3 inhibitor JSI-124 (FIG. 24illustrates STAT target gene expression) abrogates the development ofmalignant ascites in vivo (study diagram shown in FIG. 7A andalternative study diagram shown in FIG. 40). For this purpose, thePDX-model DF20 was used to assess the BLI signal after intraperitoneal(IP) injection of cancer cells and IP treatment with JSI-124 vs. vehicle(FIG. 38 illustrates IP therapy for cancer and FIG. 39 compares IP andintravenous therapy). PDX-models were injected with tumor cells andallowed to grow for 7 days, followed by 14 days of therapy with JSI-124delivered IP (FIG. 7A, top). Compared to controls, treatment withJSI-124 completely abrogated development of malignant ascites (FIG. 7B),mean day 15 log BLI signal±standard deviation, 2.19×10⁹±3.9×10⁸ vs.1.47×10⁸±6.5×10⁷, p<0.0001, two-tailed t test). Next, it was determinedwhether treatment with JSI-124 eliminates established malignant ascites.For this purpose, injected cancer cells were allowed to form malignantascites for 3 weeks prior to treatment with IP JSI-124 or vehicle (FIG.7A, bottom). Compared to vehicle treated models, significant reductionin BLI signal of malignant ascites was found (FIG. 7C, 1.6×10¹⁹±2.5×10⁹vs. 1.29×10⁹±6×10⁸, p<0.0001).

Example 8—IP Administered JSI-124 Prevents Systemic Tumor Growth andEffectively Eliminates Established Tumors

A frequently used treatment modality for advanced ovarian cancer is theintraperitoneal administration of chemotherapy, such as cisplatin, whichhas local effects, but is also systemically reabsorbed to treatpotential metastatic disease. To examine whether IP administered JSI-124prevents systemic formation/progression of solid tumor masses, DF20cancer cells were injected subcutaneously and tumors were allowed toestablish for 7 days. Tumor growth was compared following IP JSI-124treatment v. vehicle. JSI-124 was found to significantly inhibitdevelopment of tumors growth of SC tumors (FIG. 7D, 2.61×10⁹ 1×10⁹ vs.5.55×10⁸±2.2×10⁸, p=0.002) and even led to reduction of injected tumorsafter 14 days of therapy. To test the treatment of established tumorswith IP administered JSI-124, PDX models, initiating SC tumors, wereused to establish tumor for 3 weeks prior to treatment with JSI-124.Compared to vehicle, JSI-124 led to significant and near completeelimination of SC tumors (FIG. 7E, 5.65×10⁹±2.5×10⁹ vs.8.52×10⁸±3.1×10⁸, p=0.0028). However, treatment with JSI-124 resulted inincreased weight due to increased non-malignant ascites. The absence ofmalignant cells in ascites isolated from JSI-124 treated mice wasconfirmed, indicating that edema is an adverse effect.

Together, these results indicate that IP administration of JSI-124 hassignificant activity in preventing ascites formation and disruptingmalignant ascites and systemic, and therefore provides a unique approachfor the treatment of patients with ovarian cancer. The results presentedherein suggest that small molecule JAK/STAT inhibitor JSI-124, and otherJAK/STAT inhibitors, represent a feasible treatment approach forpatients with ovarian cancer.

Example 9—Ovarian Cancer Heterogeneity

A series of graphs showing ovarian cancer heterogeneity is illustratedin FIG. 19A and FIG. 19B. This experiment utilized isolated single-cellsfrom malignant effusions from a patient with highly resistant ovariancancer. In this patient, 106 single-cell transcriptomes were analyzed,and the copy number variation (CNVs) were inferred as previouslydescribed. For ovarian cancer, a large burden of CNVs were identified.While most of the CNVs were shared among cells, subpopulations wereidentified that carried unique aberrations, such as chromosome 12deletion.

Thus, this approach is helpful with reconstructing the clonalarchitecture. These patients frequently require several taps. Therefore,clonal evolution as patients undergo various treatments can be examined.

Principle component analysis (PCA) of these single-cell transcriptomesidentified a gene set that was expressed variably across cells. Apopulation of cells to the very far right expressed multiple knownmarkers of stem-like cells, such as aldehyde dehydrogenase 1A (ALDH1A),CD133, etc., among a program that now allows further evaluation of thisphenotype in other patients with ovarian cancer.

Example 10—Probing Treatment Resistance in PDX-Models

The experimental results of probing treatment resistance in patientderived xenograft (PDX)-models are shown in FIG. 20A and FIG. 20B. Theresults show 1) a predictable response; 2) non-invasive monitoring; and3) the possibility to profile minimal residual disease, single cells.

Next, the method described herein was applied to PDX models generated atDFCI/Belfer Institute. Platin-based therapies elicit predictableresponses and the PDX models allow non-invasive disease monitoring usingBLI combined with the ability to profile individual cells. The study wasa unique opportunity to probe transcriptional heterogeneity atpre-treatment, at the time of minimal residual disease, and at relapse.In the first model, DF20, mouse data nicely followed the expecteddynamics, especially for time points where single-cells were collectedfor sequencing.

Example 11—Probing MRD and Relapse at Single-Cell Resolution

The results presented herein identified a unique opportunity to probetranscriptional heterogeneity pre-treatment, at the time of minimalresidual disease, and relapse. In the first model of experiments, DF20,mice nicely followed the expected dynamics at which time pointssingle-cells were collected for sequencing. The results are presented inFIG. 21.

Example 12—Ovarian Cancer JSI-124 PDX Studies

Described herein is the study design of a PDX study and a study tointerrogate the role of this drug as an adjuvant therapy option inovarian cancer.

JSI-124 Prevention Therapy Arm

Tumors are injected intraperitoneally (IP) or subcutaneously (subQ).After day 7, mice are treated with 1 mg/kg/daily JSI-124 daily for 4weeks. The tumor volume/burden is measured by Calipers and BLI signal.SubQ tumors and spheroids are collected at the time of harvesting.Tumors are cryopreserved and snapfrozen for future studies (WB analysis,gene expression, clearance assay, etc.) A schematic is presented in FIG.25.

JSI-124 Established Tumor Therapy Arm

Tumors are injected IP or subQ. Tumors are grown for 14-21 days andtreated with 1 mg/kg/daily JSI-124 daily for 2 weeks. The tumorvolume/burden is measured by

Calipers and BLI signal. SubQ tumors and spheroids are collected at thetime of harvesting.

Tumors are cryopreserved and snapfrozen for future studies (WB analysis,gene expression, clearance assay, etc.). A schematic is presented inFIG. 26.

A schematic showing the effect of intraperitoneal, adjuvant JAK/STAT3inhibition on growth of SC tumors (DF-20) is shown in FIG. 27, while aschematic showing the effect of IV, adjuvant JAK/STAT3 inhibition ongrowth of SC tumors (DF-20) is shown in FIG. 28.

Example 13—JSI124 is Effective Against Patient-Derived Ovarian Cells ina Limited Sample Volume, Tested in a Microfluidic Device

Heterogeneous cell population isolated from abdominal ascites of ovarianpatient NACT14. A limited number of cells (˜800 cells) in a volume of 1microliter were tested for sensitivity of JSI124. On day 0, the cellswere seeded in the microfluidic device. On day 1, drug was administered,together with a blue dye (FIG. 29-FIG. 31).

Hoechst confocal imaging (stained nuclei) was used to observe cellsforty eight (48) hours after the drug was added (FIG. 32-FIG. 35).

Example 14—Online Material (Video 1-4) for Single-Cell RNA-Sequencing ofOvarian Cancer in Patients where PDX Models Reveal JAk/STAT-Inhibitionas an Effective Therapeutic Strategy to Overcome Platinum-Resistance

Described herein are patient derived spheroids treated with DMSO vs.JSI-124 for 30 or 120 minutes followed by drug removal. The resultspresented herein demonstrate invasion of DMSO-treated spheroids whileJSI-124 treated spheroids are unable to invade through the mesothelialmonolayer (Video 1 and 2).

Also described herein are spheroids generated from OVACR4 or TYKNU celllines treated with DMSO vs. JSI-124 for 30 minutes followed by drugremoval. The results demonstrate invasion of DMSO-treated spheroidswhile JSI-124 treated spheroids are unable to invade through themesothelial monolayer (Video 3 and 4).

Various modifications and variations of the described methods,pharmaceutical compositions, and kits of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific embodiments, it will be understood that it iscapable of further modifications and that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the art are intended tobe within the scope of the invention. This application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure come within known customarypractice within the art to which the invention pertains and may beapplied to the essential features herein before set forth.

What is claimed is:
 1. A method for treating or preventing agynecological tumor in a subject comprising: identifying a subject witha gynecological tumor; and administering to the subject atherapeutically effective amount of a signal transducer and activator oftranscription 3 (STAT3) activity inhibitor, thereby treating orpreventing the gynecological tumor in the subject.
 2. The method ofclaim 1, wherein the gynecological tumor comprises an ovarian tumor. 3.The method of claim 1, wherein the subject is identified as havingelevated STAT3 activity, or wherein the subject is identified as in needof inhibiting STAT3 activity.
 4. The method of claim 1, wherein theSTAT3 activity inhibitor is administered intraperitoneally.
 5. Themethod of claim 1, wherein the STAT3 activity is selected from the groupconsisting of STAT3 phosphorylation, STAT3 dimerization, STAT3 bindingto a polynucleotide comprising a STAT3 binding site, STAT3 binding togenomic DNA, activation of a STAT3 responsive gene and STAT3 nucleartranslocation.
 6. The method of claim 1, wherein the STAT3 inhibitorcomprises pyrimethamine, atovaquone, pimozide, guanabenz acetate,alprenolol hydrochloride, nifuroxazide, solanine alpha, fluoxetinehydrochloride, ifosfamide, pyrvinium pamoate, moricizine hydrochloride,3,3′-oxybis[tetrahydrothiophene, 1,1,1′,1′-tetraoxide],3-(1,3-benzodioxol-5-yl)-1,6-dimethyl-pyrimido[5,4-e]-1,2,4-triazine-5,7(-1H,6H)-dione,2-(1,8-Naphthyridin-2-yl)phenol,3-(2-hydroxyphenyl)-3-phenyl-N,N-dipropylpropanamide as well as anyderivatives of these compounds or analogues thereof.
 7. The method ofclaim 1, wherein the STAT3 activity inhibitor comprises JSI-124(cucurbitacin I).
 8. The method of claim 7, wherein the JSI-124(cucurbitacin I) is administered at a dose of about 0.1 μM.
 9. Themethod of claim 7, wherein tumor cell growth in the subject's abdomen isinhibited.
 10. The method of claim 7, wherein subcutaneous tumor cellgrowth in the subject is inhibited.
 11. The method of claim 7, whereinovarian tumor cell metastases in the subject are inhibited.
 12. Themethod of claim 7, wherein malignant abdominal fluid (ascites) isinhibited.
 13. The method of claim 7, wherein tumor recurrence isprevented.
 14. The method of claim 1, further comprising administering atherapeutically effective amount of a chemotherapeutic agent.
 15. Themethod of claim 14, wherein the chemotherapeutic agent comprises aplatinum-based chemotherapeutic agent or a taxane-based chemotherapeuticagent.
 16. The method of claim 15, wherein the platinum-basedchemotherapeutic agent comprises cisplatin or carboplatin.
 17. Themethod of claim 14, wherein the chemotherapeutic agent is administeredprior to, simultaneously with, or subsequent to administration of theSTAT3 activity inhibitor.
 18. The method of claim 1, wherein the subjectis a human.
 19. The method of claim 1, wherein the subject has receivedprior treatment for the gynecological tumor.
 20. The method of claim 1,wherein the gynecological tumor is resistant to platinum-basedchemotherapy.
 21. The method of claim 1, wherein the subject has minimalresidual disease (MRD) following platinum-based chemotherapy.
 22. Amethod for treating a gynecological tumor in a subject comprising:identifying a subject with a gynecological tumor; administering to thesubject a therapeutically effective amount of a chemotherapeutic agentto inhibit the gynecological tumor; and administering to the subject atherapeutically effective amount of a STAT3 activity inhibitor toprevent recurrence of the gynecological tumor and development ofmetastases, thereby treating or preventing the gynecological tumor inthe subject.
 23. The method of claim 22, wherein the STAT3 activityinhibitor is administered at least one month after administration of thechemotherapeutic agent.
 24. A method for treating a platinum-resistantgynecological tumor in a subject comprising: identifying a subject witha platinum-resistant gynecological tumor; administering to the subject atherapeutically effective amount of a STAT3 activity inhibitor;administering to the subject a therapeutically effective amount of achemotherapeutic agent, thereby treating the platinum-resistantgynecological tumor in the subject.
 25. The method of claim 19, whereinthe STAT3 activity inhibitor is administered prior to thechemotherapeutic agent.
 26. A method of treating platinum-basedchemotherapy resistant ovarian cancer comprising treating a subject inneed thereof with an inhibitor of the JAK/STAT pathway.
 27. The methodaccording to claim 26, wherein the subject has minimal residue disease(MRD) or the ovarian cancer is a relapse.
 28. The method according toclaim 26 or 27, wherein the inhibitor is JSI-124.
 29. A method oftreating ovarian cancer comprising treating a subject in need thereofwith an agent capable of inhibiting expression or activity of one ormore genes or polypeptides selected from the group consisting of JAK1,STAT3, STAT2, STAT1, OSMR, STAT6, RELA, ERBB2, GF1R, ERBB3, IL10RB,FGFR1, CXCR4, CXCL2, CXCL10, CXCL11, HLA-DRA, HLA-DRB1, HLA-DQB1, IDO1,MYBL2 and FGF13.
 30. The method according to claim 29, wherein theovarian cancer is chemotherapy resistant.
 31. The method according toclaim 30, wherein the ovarian cancer is platinum-based chemotherapyresistant.
 32. The method according to any of claims 29 to 31, whereinsaid agent comprises a therapeutic antibody, bi-specific antibody,antibody fragment, antibody-like protein scaffold, aptamer, geneticmodifying agent or small molecule.
 33. The method according to any ofclaims 29 to 32, wherein the gene or polypeptide is a surface orsecreted gene.
 34. The method according to claim 33, wherein the agenttargets a secreted protein or a receptor for the secreted protein. 35.The method according to claim 33, wherein the agent targets a ligand fora surface receptor.
 36. The method according to any of claims 29 to 35,further comprising administering platinum-based chemotherapy.
 37. Amethod of predicting a response to platinum-based chemotherapy in asubject suffering from ovarian cancer comprising detecting in a tumorsample obtained from the subject expression of one or more genesselected from the group consisting of JAK1, STAT3, STAT2, STAT1, OSMR,STAT6, RELA, ERBB2, GF1R, ERBB3, IL10RB, FGFR1, CXCR4, CXCL2, CXCL10,CXCL11, HLA-DRA, HLA-DRB1, HLA-DQB1, IDO1, MYBL2 and FGF13, wherein highexpression in the tumor sample indicates a weak response toplatinum-based chemotherapy and low expression indicates a strongresponse to platinum-based chemotherapy.
 38. A method of detectingovarian cancer stem cells in a tumor sample obtained from a subjectsuffering from ovarian cancer comprising detecting expression of a genesignature comprising one or more genes selected from the groupconsisting of ALDH1A3, CD24, CD133, FN1, ACTA2, MYL9, GAS6, IGFBP5,FGFR1, CALD1, CFI, CRB2, PRDX4, IGFBP6 and RPS20.
 39. A method oftreating ovarian cancer comprising treating a subject in need thereofwith an agent capable of targeting ovarian cancer stem cellscharacterized by a gene signature comprising one or more genes selectedfrom the group consisting of ALDH1A3, CD24, CD133, FN1, ACTA2, MYL9,GAS6, IGFBP5, FGFR1, CALD1, CFI, CRB2, PRDX4, IGFBP6 and RPS20.
 40. Themethod according to claim 39, wherein the agent targets a surfaceprotein on the ovarian cancer stem cells.
 41. The method according toclaim 40, wherein the agent comprises a therapeutic antibody, bispecificantibody, antibody fragment, antibody-like protein scaffold, aptamer orCAR T cell.
 42. The method according to any of claims 39 to 41, furthercomprising administering platinum-based chemotherapy.
 43. The method oftreatment according to any of claims 26 to 35 or 39 to 41, wherein thetreatment is administered as an adjuvant or neoadjuvant therapy.
 44. Themethod of claim 1, wherein the inhibitor comprises a therapeuticantibody, bi-specific antibody, antibody fragment, antibody-like proteinscaffold, aptamer, genetic modifying agent or small molecule.